Download Hi-Res ImageDownload to MS-PowerPointCite This:Biochemistry 2017, 56, 44, 5886-5899

ArticleOctober 11, 2017

Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N-Termini
Click to copy article linkArticle link copied!

Purva P. Bhojane
Purva P. Bhojane
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, United States
More by Purva P. Bhojane
Michael R. Duff Jr.
Michael R. Duff, Jr.
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, United States
More by Michael R. Duff, Jr.
Khushboo Bafna
Khushboo Bafna
Genome Science and Technology Program, University of Tennessee, Knoxville, Tennessee 37996-0840, United States
More by Khushboo Bafna
Pratul Agarwal
Pratul Agarwal
Computer Science and Mathematics Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States
More by Pratul Agarwal
http://orcid.org/0000-0002-3848-9492
Christopher Stanley
Christopher Stanley
Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States
More by Christopher Stanley
http://orcid.org/0000-0002-4226-7710
Elizabeth E. Howell*
Elizabeth E. Howell
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, United States
Genome Science and Technology Program, University of Tennessee, Knoxville, Tennessee 37996-0840, United States
*Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996-0840. Phone: 865-974-4507. Fax: 865-974-6306. E-mail: [email protected]
More by Elizabeth E. Howell
http://orcid.org/0000-0001-6157-433X

Open PDF Supporting Information (3)

Biochemistry

Cite this: Biochemistry 2017, 56, 44, 5886–5899

Click to copy citationCitation copied!

https://doi.org/10.1021/acs.biochem.7b00822

Published October 11, 2017

Request reuse permissions

Abstract

Click to copy section linkSection link copied!

R67 dihydrofolate reductase (DHFR) is a homotetramer with a single active site pore and no sequence or structural homology with chromosomal DHFRs. The R67 enzyme provides resistance to trimethoprim, an active site-directed inhibitor of Escherichia coli DHFR. Sixteen to twenty N-terminal amino acids are intrinsically disordered in the R67 dimer crystal structure. Chymotrypsin cleavage of 16 N-terminal residues results in an active enzyme with a decreased stability. The space sampled by the disordered N-termini of R67 DHFR was investigated using small angle neutron scattering. From a combined analysis using molecular dynamics and the program SASSIE (http://www.smallangles.net/sassie/SASSIE_HOME.html), the apoenzyme displays a radius of gyration (R_g) of 21.46 ± 0.50 Å. Addition of glycine betaine, an osmolyte, does not result in folding of the termini as the R_g increases slightly to 22.78 ± 0.87 Å. SASSIE fits of the latter SANS data indicate that the disordered N-termini sample larger regions of space and remain disordered, suggesting they might function as entropic bristles. Pressure perturbation calorimetry also indicated that the volume of R67 DHFR increases upon addition of 10% betaine and decreased at 20% betaine because of the dehydration of the protein. Studies of the hydration of full-length R67 DHFR in the presence of the osmolytes betaine and dimethyl sulfoxide find around 1250 water molecules hydrating the protein. Similar studies with truncated R67 DHFR yield around 400 water molecules hydrating the protein in the presence of betaine. The difference of ∼900 waters indicates the N-termini are well-hydrated.

Subjects

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as a cofactor. THF and its derivatives serve as cellular cofactors for one-carbon transfer reactions involved in the synthesis of nucleotides such as purines and thymidine, amino acids such as methionine and glycine, and various other metabolites. Trimethoprim is a potent inhibitor of Escherichia coli chromosomal DHFR (EcDHFR) and has been widely used as an antibacterial drug. The gene encoding R67 DHFR, carried by an R-plasmid, confers resistance against trimethoprim. Recent clinical isolates of E. coli causing urinary tract infections have the gene encoded in class I integrons flanked by other drug resistance genes. (1) There are no antibiotics that target R67 DHFR, though promising leads have recently been discovered. (2,3) This type II DHFR (78 amino acids long) is genetically and structurally unrelated to EcDHFR. R67 DHFR is a homotetramer, and each monomer has five antiparallel β-strands that assemble into a dimer with a six-stranded β-barrel at the subunit interface. Using loop–loop interactions, two dimers assemble into a tetrameric “doughnut” with a single active site pore. (4)

Numerous experiments indicate the 16–20 N-terminal residues of R67 DHFR are disordered and can tolerate various sequences. For example, several disorder predictors indicate the N-terminal sequence is intrinsically disordered. (5) Also, the first 17 amino acids for each monomer do not appear in the dimer crystal structure. (6) The N-termini can be cleaved after F16 by chymotrypsin treatment, and the truncated protein is almost fully active, although 2.6 kcal/mol less stable. (7) The truncated tetrameric protein was crystallized, and the structure was first determined at a resolution of 1.7 Å (4) and later at 0.96–1.26 Å. (8,9) High thermal factors in the latter structure suggest the stretch of residues 17–21 is also disordered. In addition, electron densities for residues 21–23 were diffuse, indicating high mobility. (9)

Other type II DHFR variants (e.g., R388 and R751) show different N-terminal sequences, but the same core sequence contributes to the β-barrel structure. (5,10,11) This can also be seen from a sequence alignment of the type II DHFR variants (12) showing non-identity in the first 21 residues. His tags can also be added to the N-termini. (13−15) In addition, a tandem array of four R67 DHFR gene copies encodes a protein in which the C- and N-termini of the first and second monomers are fused as well as the second and third monomers and the third and fourth monomers. The resulting Quad1 protein possessing 4 times the molecular mass of the R67 DHFR monomer is stable as well as functional. (16) Asymmetric mutations in the core of R67 DHFR that favor one topology (17,18) are used in these Quad constructs. Similarly, the N-terminal sequences from R388 and R751 can be used as the linker domains to give a functional monomeric Quad4 protein. (5) These various experiments and constructs indicate the N-termini can be fused without a loss of function. Pelletier and co-workers have made similar dimeric fused constructs of R67 DHFR. (19,20)

To gain information about the conformational space occupied by the disordered N-terminal sequences in R67 DHFR, we used small angle neutron scattering (SANS) experiments. Because of the inherent contrast between hydrogen and deuterium atoms, SANS data of the hydrogenated protein in D₂O buffer allow modeling of the ensemble of conformations sampled by the disordered tails.

As disordered sequences often undergo coupled binding and folding, we monitored any potential changes in the conformational sampling of the disordered tails upon formation of a binary complex (R67 DHFR–NADP⁺) or a ternary complex (R67 DHFR–NADP⁺–DHF). Also, osmolytes have been shown to exert protein-stabilizing forces via a preferential exclusion mechanism. (21,22) To determine whether addition of an osmolyte leads to folding of the termini, we added deuterated betaine to examine any changes in the R67 DHFR shape using SANS.

Also of interest, the water associated with the protein surface comprises the hydration layer, which can be differentiated from the bulk solvent. The first hydration shell can contain tightly bound water as well as water that can freely exchange. These differences are due to the varied environments associated with the protein surface, which can display different clefts and bumps as well as different atom types. (23) Computational studies have shown that water molecules hydrating the disordered chains exhibit properties different from the properties of those surrounding globular domains, in terms of both the number of waters and the structural order of the water molecules in the hydration layer. (24,25)

To monitor the preferential hydration of full-length and truncated R67 DHFR, we used hydrogenated osmolytes in a D₂O buffer solution in additional SANS experiments. This is analogous to a H₂O/D₂O contrast variation approach; i.e., the contrast created by addition of a hydrogenated osmolyte allows measurement of the hydration shell associated with R67 DHFR. The contrast created by osmolytes differentiates between the hydration layer and the bulk solvent. The information obtained from the scattering contrast can be used to obtain the number of water molecules in the hydration layer that are responsible for exclusion of the added osmolyte from the protein surface.

Methods

Click to copy section linkSection link copied!

Protein Expression and Purification

Full-length R67 DHFR (MIRSSNEVSN PVAGNFVFPS NATFGMGDRV RKKSGAAWQG QIVGWYCTNL TPEGYAVESE AHPGSVQIYP VAALERIN) was expressed and purified as described by Reece et al. (7) Briefly, cell lysates were subjected to ammonium sulfate precipitation and ion-exchange column chromatography to purify the protein to homogeneity. Purified samples were dialyzed against distilled, deionized H₂O and lyophilized.

Chymotrypsin-truncated R67 DHFR was obtained as described by Reece et al., (7) starting from full-length His-tagged R67 DHFR. The His-tagged construct has the synthetic R67 DHFR gene (7) cloned into the pRSETB vector from Invitrogen. (26) Purification was performed with a nickel-nitrilotriacetic acid (Ni-NTA) column (Qiagen), followed by elution from a DEAE fractogel column. The resulting protein was incubated with immobilized chymotrypsin (Sigma-Aldrich) in 10 mM Tris/1 mM EDTA, pH 8.0 buffer overnight at 4 °C and later at room temperature for ≤24 h. Chymotrypsin cleaves after F16 in the R67 DHFR sequence (or after F47 in the His-tagged sequence). The progress of the reaction was monitored by sodium dodecyl sulfate electrophoresis (see Figure S1). Immobilized chymotrypsin was removed, and the truncated tetramer was separated from peptide fragments by gel filtration at pH 8 using G75 Sephadex. A Ni-NTA column further separated the cleaved N-terminus from the tetrameric core of the protein. The purified truncated R67 DHFR was dialyzed against water using a 7 kDa cutoff membrane and then lyophilized. Protein concentrations were determined by measuring the absorbance at 280 nm of the solution using an extinction coefficient determined with a bicinchoninic acid (Pierce) assay.

Small Angle Neutron Scattering (SANS)

The sample of lyophilized, full-length, apo R67 DHFR was reconstituted in 20 mM deuterated Tris buffer in D₂O (pD 7.5). Experiments were also performed to study any changes in the ordering of the N-termini upon binding of NADP⁺ to apo R67 DHFR (binary complex formation) and upon binding of dihydrofolate (DHF) to the R67 DHFR–NADP⁺ complex (ternary complex formation) under saturating ligand concentrations (3 mM NADP⁺ for binary samples and 3 mM NADP⁺ and 2 mM DHF for ternary samples). To study the effect of betaine on the disordered N-termini of R67 DHFR, the change in overall shape and compaction of apoprotein in the presence of 20% deuterated betaine was explored. Additionally, samples of full-length apo R67 DHFR with no osmolyte and with the osmolytes betaine and dimethyl sulfoxide (DMSO) were prepared to investigate protein hydration. The osmolytes were hydrogenated to create a contrast with the deuterated buffer conditions, allowing measurement of changes in preferential hydration of apo R67 DHFR. (27) The concentrations of osmolytes ranged from 2.5 to 20% (w/v) for betaine and from 2.5 to 17.5% (v/v) for DMSO. The protein concentrations ranged from 4.5 to 7.5 mg/mL. Similar sample sets using the truncated R67 DHFR protein with 0 to 20% (w/v) betaine were prepared. The concentration of the truncated protein was 2.4–2.6 mg/mL. Buffer controls were run to detect the background scattering. All samples were prepared, centrifuged, and loaded into banjo-shaped quartz cuvettes (Hellma USA, Plainville, NY) with a path length of 2 mm.

Experiments were performed on the EQ-SANS instrument at the Spallation Neutron Source at the Oak Ridge National Laboratory. In 60 Hz operation mode, a 4 m sample–detector distance with a 2.5–6.1 Å wavelength band was used. Neutron exposure times were approximately 1 h, and the scattered neutrons were detected on a 1 m × 1 m two-dimensional detector at 25 °C.

The data collected for all experiments were reduced using MANTID Plot, (28) and the total two-dimensional scattering was corrected by the scattering from the empty quartz cell. Then, the scattering was normalized by the incident beam flux and radially averaged to obtain the absolute scale intensity, I(q), versus scattering angle, q. The background scattering for the respective buffers was subtracted from the total scattering. Guinier analysis with a linear plot of ln I(q) versus q² for low-q data gave a slope of −(R_g²)/3, where R_g is the radius of gyration and the intercept on the Y-axis gave the I(0) value. Estimates of R_g and zero-angle scattering intensity I(0) were obtained using eq 1: (27)

(1)

where I(q) is the scattering intensity at small angles (q).

The data were also analyzed using GNOM in the ATSAS package. (29) GNOM reads the scattering profile and evaluates the particle distance distribution function, P(R), in a defined range of distances and yields the apparent radius of gyration (R_g) and zero-angle scattering intensity I(0). Data for each sample were fit using Guinier analysis and GNOM (see Table S1).

The R_g values and zero-angle scattering intensities, I(0), of R67 DHFR in the presence of varying concentrations of osmolytes (betaine and DMSO) were normalized by the protein concentration of each sample. To obtain information about the preferential hydration of R67 DHFR and the effect of osmolytes on hydration, the change in I(0) with an increasing concentration of osmolytes obtained from the GNOM analysis was fit to eq 2 from ref (27)

(2)

where I_s(0) and I(0) are the zero-angle scattering intensities in the presence and absence of an osmolyte, respectively, f_v, or fractional volume, is the concentration of osmolyte added (w/v for betaine and v/v for DMSO), ρ_w, ρ_s, and ρ_p are the scattering-length densities of water, solute (=osmolyte), and protein, respectively, and V_p and V_w are the volumes of protein and protein-associated water, respectively. The scattering-length densities of the full-length (3.248 × 10¹⁰ cm^–2) and truncated (3.228 × 10¹⁰ cm^–2) proteins, betaine (0.817 × 10¹⁰ cm^–2), and DMSO (−0.051 × 10¹⁰ cm^–2) and the protein volumes were calculated using the online tool MULCh. (30) The volume of protein-associated water gives the number of water molecules in the hydration layer of R67 DHFR upon addition of the osmolyte. Note that eq 2 assumes V_p and V_w remain constant across the range of osmolyte concentrations used. In other words, the measured V_w is an average across the betaine range used for the preferential hydration SANS experiments.

Analysis Using MD and SASSIE

Our next step was to analyze the SANS data using models generated via MD and SASSIE (see http://www.smallangles.net/sassie/SASSIE_HOME.html). (31) The latter creates atomistic models of the protein using Monte Carlo simulations, calculates theoretical scattering data for these models using SasCalc or Xtal2SAS tools, and compares the theoretical data to the experimental data. The experimental SANS data were interpolated into SASSIE in a defined q range using the data interpolation module.

As both MD and SASSIE require a model of the full-length protein sequence to generate structures for fitting, sixteen residues (MIRSSNEVSNPVAGNF-) were added to each of the four N-termini of the truncated R67 DHFR structure (PDB entry 2RH2) (8) using Modeller (version 9.15). (32) A total of 100 models were generated, and 10 structures with the lowest discrete optimized potential energy score were used further. The selected models were minimized under vacuum for 10000 steps. The minimized structures were then solvated in a SPC/E water box (33) and equilibrated using the protocol as described by Ramanathan et al. (34) The protein and ligand parameters were generated using AMBER force field ff14SB. Extensive MD simulations were performed for the apoenzyme, the binary complex with NADPH, and the ternary complex with NADPH and DHF using the AMBER 14 simulation package. (35) Initially, 10 models of the apoenzyme were simulated for 100 ns each, and selected conformations from these runs that gave good fits to the SANS data were further studied for four apo simulations of 1 μs each. Therefore, the total aggregate sampling for apo systems was 5 μs. Similarly, nine MD simulations for the binary complex and five for the ternary complex were performed for 1 μs each, providing aggregate 9 μs and 5 μs MD sampling for binary and ternary complexes, respectively.

Additionally, two more models were built. The first model had two pairs of N-termini interacting with each other on both sides of the pore, and a second model moved all the four termini to block access to the active site pore. This approach allowed the construction of numerous structures in which the N-termini sample a large area of conformational space.

Frames from the MD trajectories were analyzed in SASSIE using the SasCalc module that generated theoretical SANS profiles, which were then compared to the experimental SANS data using the χ² analysis module. Those structures with a low χ² value (<10) were chosen as good fits. Complex Monte Carlo simulations also generated additional conformers for fitting. In this process, the core of the protein remained constant, and only alternate conformations of the 21 N-terminal amino acids were generated. Acceptable frames avoided atom overlaps. In addition, on the basis of the average R_g obtained (for example, ∼21.5 Å for apo R67 DHFR), directed Monte Carlo sampling additionally generated >100000 structures with R_g values limited to a range from 20.5 to 22.5 Å. These structures were subjected to a 500-step minimization using NAMD. Again, the theoretical SANS profiles were calculated using the SasCalc module in SASSIE, followed by a χ² analysis. We chose to use a strategy by which we analyzed single structures, as opposed to an ensemble structure method, because our relatively exhaustive analysis in SASSIE using our MD and Monte Carlo structures was found to be adequate to fit our SANS data.

Both MD and SASSIE analyses were performed to fit the experimental SANS profiles for the ligand-bound complexes (binary and ternary) as well as for apo R67 DHFR in 20% deuterated betaine. To generate sufficient conformers with low χ² values, the apo, binary, and ternary structures were interconverted by a Python script by adding or removing the coordinates of the NADP⁺ and DHF ligands in the active site pore of the MD and Monte Carlo-generated structures. The ligand coordinates were obtained from PDB entries 2RK1 and 2RK2. (8) For the analysis of the binary data, initially one NADP⁺ was positioned in the active site pore as per the 2RK2 crystal structure; (8) however, only 92 good fits were obtained. To gain more fits, we considered the possibility that a second cofactor could bind as the concentration of NADP⁺ in the SANS sample was high (3 mM) and the active site pore can accommodate two homoligands. (36) There are four sets of coordinates for NADP⁺ in the 2RK2 crystal structure because of the symmetry of the active site. Therefore, we positioned a second set of NADP⁺ coordinates in a symmetry-related position using the 2RK2 structure and continued with the analysis. A workflow and summary of the various steps in our analyses for apo, binary, and ternary complexes are provided in Figure S2.

Snapshots from MD simulations were also used to compute the number of water molecules present in the first solvation shell of the protein. AMBER’s ptraj module and command watershell, with the default cutoff of 3.4 Å, were used.

Data Mining

The structures that best fit the SANS profiles were data mined to find the most frequent interactions between the N-terminus and all the other residues in the protein. To analyze the structures, the cpptraj program in Amber14 (35) was used to calculate the distances between the center of mass (COM) of each residue and all the other individual residues in the R67 DHFR tetramer. A python script was then used to determine the minimum distance between the COM for each pair of residues for all structures that fit the SANS data. Additionally, the number of times the center of masses for each pair of residues was within 5 Å was calculated. Heat maps of the inter-residue interactions were created from the matrix of residue pair interactions using Matlab (version r2017a).

Differential Scanning Calorimetry (DSC)

Thermal unfolding of full-length and truncated R67 DHFRs was monitored between 25 and 95 °C using a Microcal VP differential scanning microcalorimeter. The concentration of full-length R67 DHFR was 150–160 μM in MTA buffer (100 mM MES, 50 mM Tris, and 50 mM acetic acid) at pH 8. Samples were also prepared in MTA buffer with 20% betaine or 15% DMSO. Scans were repeated two times with scan rates of 1 °C/min. For truncated R67 DHFR, concentrations of 50–100 μM were used with a scan rate of 1 °C/min. The data obtained were analyzed using Origin version 7.0 supplied by the manufacturer and the melting temperatures obtained.

Pressure Perturbation Calorimetry (PPC)

Effects of betaine on the volume, or hydration, of R67 DHFR were estimated using the change in the thermal expansion coefficients (α) of the protein in the absence and presence of betaine. Pressure perturbation calorimetry (PPC) can be used to determine the α_o value in buffer containing osmolytes using the following equation (eq 3): (37,38)

(3)

where α_s and α_o are the thermal expansion coefficients for the solute and solvent, respectively, ΔQ is the heat released or absorbed after each application, or release, of pressure, T is the temperature, m_s is the mass of the solute in the solution, V_s is the specific volume of the solute, and Δp is the change in pressure applied above the solution. A VP-DSC instrument from MicroCal (Malvern) outfitted with a PPC appendage was used to calculate the α_s for R67 DHFR. Pressure pulses of 60 psi of nitrogen were applied above the sample, using buffer alone as a reference. The thermal expansion coefficients were determined between 10 and 95 °C in 2.5 °C increments. Samples of 3–5 mg/mL R67 DHFR (these concentrations are equivalent to 90–150 μM for full-length and 99–150 μM for truncated R67 DHFRs) were prepared in 45 mM Na₂HPO₄, pH 8.0 buffer containing 0, 10, or 20% (w/w) betaine. Control experiments using buffer versus buffer, buffer versus water, and water versus water were used to correct the α_s of R67 DHFR for the thermal expansion of the buffer and water components of the sample (which are contained in α_o). The raw data from the PPC were manually curated so that they could be integrated using NITPIC. (39) Files with the heat obtained from NITPIC were used to analyze the PPC data in the Origin 7.0 software package provided by MicroCal. The mass of the solute in the solution was determined spectroscopically, and the specific volume of 0.716 mL/g for R67 DHFR was previously obtained. (40)

Results

Click to copy section linkSection link copied!

SANS of Apo R67 DHFR

Representative SANS profiles for full-length and truncated apo R67 DHFR are shown in Figure 1A and Figure S3A, respectively (see the Supporting Information). A dimensionless Kratky plot (41) of the full-length and truncated proteins indicates that both are globular (Figure S3C). Analysis using GNOM yields an R_g value of 21.89 ± 0.12 Å for the full-length protein (see Figure 1B) and 17.86 ± 0.14 Å for truncated R67 DHFR (Figure S3B). The latter is comparable to the R_g values of 17.1 and 17.5 Å for the 2RH2 (8) and 2GQV (9) crystal structures of truncated R67 DHFR, respectively, calculated using CRYSON. (42) The molecular weight of R67 DHFR was calculated from the I(0) and R_g of the SANS profile, using a model that is independent of protein concentration. (43) A value of 36470 g/mol matches well with the expected value of 33720 g/mol for full-length R67 DHFR, indicating that the sample is not aggregating under our conditions (Table 1).

Figure 1. SANS profile and GNOM analysis for apo, full-length R67 DHFR. (A) Normalized scattering intensity of the protein, I(q)/I(0), with an increase in q. The SANS profile was obtained by subtracting the scattering contribution from the buffer and normalizing the scattering intensity by I(0). (B) A GNOM fit of the profile gave the pairwise distance distribution and an R_g value of 21.89 ± 0.12 Å.

Table 1. Data Analysis of the SANS Profiles and Comparison of R_g Values for R67 DHFR Obtained Using GNOM and SASSIE Analyses

					no. of good fits in SASSIE (χ² < 10)
protein samples	I(0)	theoretical MW (Da)	calculated MW (Da)	GNOM R_g (Å)	no.	R_g range (Å)	mean R_g (Å)
truncated R67 DHFR	0.0227	26906	23390	17.86 ± 0.14	–	–	–
apo R67 DHFR	0.1711	33720	36470	21.89 ± 0.12	7936	20.84–23.53	21.46 ± 0.50
R67 DHFR–2NADP⁺	0.1633	35218	33630	21.45 ± 0.14	758	20.67–22.77	21.56 ± 0.39
R67 DHFR–NADP⁺–DHF	0.1605	34912	35065	21.45 ± 0.18	15551	20.14–22.74	20.64 ± 0.27
apo R67 DHFR in 20% deuterated betaine	0.1148	33720	33620	23.08 ± 0.12	58277	21.04–25.94	22.78 ± 0.87

As described in Methods, we used MD and the NIST program SASSIE to gain information about the space sampled by the N-termini. We generated 19 μs of MD trajectories and ∼307000 structures from nondirected as well as directed Monte Carlo analysis in SASSIE. A large number of structures were used to analyze our SANS data, and the volumes sampled by the N-termini are shown in Figure S4, differentiated by the method by which they were generated (e.g., MD, Monte Carlo, or hand built). The directed Monte Carlo analysis restricted structures to an R_g range of 20.5–22.5 Å, substantially helping us find conformers that fit the SANS data. Figure 2A shows a χ² versus R_g plot for the 117000 frames from the directed Monte Carlo simulations and the apo frames obtained from MD runs for binary and ternary complexes upon removal of the ligands using a Python script. The χ² versus R_g plot shows a “U” shape, indicating neither very compact nor very extended states fit the data well. Instead, more intermediate structures fit the data. We identified 7936 structures that fit the apoenzyme SANS data with a χ² value of <10. The lowest χ² value obtained was 1.8, and the R_g of the corresponding frame was 22.24 Å. An average R_g value of 21.46 ± 0.50 Å was obtained (see Table 1), which is similar to the R_g value obtained from the GNOM fitting.

Figure 2. SASSIE analysis of apo R67 DHFR suggests compaction of the N-termini. (A) Analysis of the frames generated by directed Monte Carlo sampling (117000, black filled circles) and frames from the MD runs (154000, cyan filled circles). The red line indicates a χ² = 10 cutoff. Data with χ² > 50 are not shown for the sake of clarity. (B) Overlays of the theoretical SANS profiles for the best [χ² = 1.8 (red squares)] and worst [χ² = 430 (blue triangles)] fits compared to the experimental SANS data (black circles). (C) Corresponding best and worst structures. (D) Density plot describing all the space sampled by MD and Monte Carlo structures (dark gray mesh) representing most of the available “structural space”. The structures identified by SASSIE as providing good fits to the experimental SANS data are shown by green mesh. One of the good models describing full-length homotetrameric R67 DHFR is shown in the center of the mesh.

SASSIE also generates mesh plots that show the space sampled by the 21 N-terminal residues. All ∼461000 structures (307000 from Monte Carlo and 154000 from MD) sample the area shown by the dark gray mesh in Figure 2D. Best fits identified by SASSIE show a more restricted area explored by the N-termini (see the green mesh in Figure 2D), indicating compaction of the N-termini as compared to full extension. This trend is also identified in a plot of the center of mass for the N-terminal methionines (see Figure S5 for those structures for which χ² < 10). Here the tendency of the methionines to sample space mostly near the sides of the protein core can be seen. However, other methionine positions fit the data, indicating other successful sampling positions. The range of 20.84–23.53 Å (see Table 1) for the best fit R_g values generated by SASSIE also indicates sampling of compact and slightly extended conformations of the N-termini. Any asymmetry in the mesh and sampling positions likely arises from nonconvergence of the MD trajectories, even though a total of 19 μs was used. In contrast, the protein termini have millisecond to second sampling times available.

Effect of Ligand Binding on the Disordered Termini in R67 DHFR

SANS data were also collected to monitor if there were any changes in the disordered N-termini of R67 DHFR upon ligand binding. Data collected for binary (R67 DHFR–NADP⁺) and ternary (R67 DHFR–NADP⁺–DHF) complexes were analyzed using GNOM. A comparison of the pairwise distribution plots for the apo, binary, and ternary complexes is shown in Figure 3A. The R_g values for the apoprotein, NADP⁺ binary complex, and NADP⁺–DHF ternary complex are 21.89 ± 0.12, 21.45 ± 0.14, and 21.45 ± 0.18 Å, respectively.

Figure 3. SASSIE analysis of ligand-bound R67 DHFR complexes indicates compaction of the N-termini. (A) GNOM fits of the pairwise distribution plots of R_g for apo R67 DHFR (green line), R67 DHFR–NADP⁺ binary (dashed orange line), and R67 DHFR–NADP⁺–DHF ternary (dotted blue line) complexes. SANS data were collected using 6.05 mg/mL DHFR in 20 mM deuterated Tris buffer in D₂O (pD 7.0) with no osmolyte. Binary or ternary complexes were formed by adding 3 mM NADP⁺ or NADP⁺ with 2 mM DHF, respectively. The R_g values for the apoprotein, binary complex, and ternary complex are 21.89 ± 0.12, 21.45 ± 0.14, and 21.45 ± 0.18 Å, respectively. While it may seem that the maximal diameter of the protein (D_max) varies, it is more that there is not a well-defined D_max value for proteins with flexible regions. Rather, a small range of D_max values all appear satisfactory. Within this “optimized” range, R_g and I(0) values are not changing significantly, allowing reliable parameters to be gained from the analysis. (B) Comparison of the mesh plots obtained from SASSIE for the apo (green) and NADP⁺–DHF ternary (blue) complexes. Bound DHF (cyan) and NADPH (magenta) are shown as ball-and-stick models in the center of the active site pore. (C) Venn diagram comparing the overlaps associated with the number of apo, binary, and ternary best fits. Figure S6 shows similar figures for the binary complex.

To gain deeper insights into the disordered tail conformations, SANS data for the R67 DHFR–NADP⁺ binary and R67 DHFR–NADP⁺–DHF ternary complexes were further analyzed using both MD and SASSIE. SASSIE analyses used the same set of ∼307000 frames described above for the apoenzyme, with ligands added by a Python script with 154000 frames from MD simulations. Figure S2 indicates the various steps used to generate best fit conformers. The plots shown in Figures S6 and S7 are for the analyses of binary and ternary data, respectively. Fitting our data to structures lacking the ligands did not yield any conformers with good χ² values. Therefore, we repeated our analyses with ligands in the protein structures. Our first fits to a single bound NADP⁺ yielded only 92 conformers with good χ² values, so we docked in another cofactor as two homoligands can bind in the active site pore. (36) This analysis yielded a total of 758 frames with χ² values of <10 for the binary data. The number of frames that fit the binary data is low, suggesting (1) a mixed population of species may be present (i.e., both singly and doubly bound NADP⁺) and (2) our model of the 2NADP⁺ complex may only approximate this species. Fitting of the ternary complex data was more successful, yielding 15551 frames with a χ² of <10. The best χ² values for the binary and ternary complexes were 5.2 and 4.8, respectively. The mean R_g value obtained for the 758 good binary structures was 21.56 ± 0.39 Å, which is within the error of the R_g obtained by GNOM analysis. However, the mean R_g value for 15551 structures for the ternary complex was 20.64 ± 0.27 Å, which is different from the R_g value determined by GNOM analysis. The range of the R_g values in the acceptable SASSIE fits for the ternary complex was 20.14–22.74 Å, which is slightly lower than the range obtained for the apoprotein.

A comparison of density plots (or space sampled) in Figure 3B shows the N-termini of the apoprotein and ternary complex sample space at the monomer–monomer interfaces at the sides of the protein. In addition, 28% of the good fits for the ternary data fit to the apo data, indicating overlap in the conformations sampled by the N-termini in the apo and ternary complex. This also can be noted from the Venn diagram shown in Figure 3C. Also, all the 758 frames that fit to the binary data fit the apo data, suggesting similar conformational sampling of the N-termini under both conditions.

A COM for the N-terminal methionine residues in the best fit frames of the ternary complex data was again calculated. These values, represented in Figure S7E, depict sampling of a restricted space near the sides of the protein (i.e., monomer–monomer interface). A comparison of the mesh plots for the apo form and binary complex is additionally shown in Figure S6D, and a COM representation for the 758 frames for the binary analysis is shown in Figure S6F.

Effect of Betaine on the R67 DHFR Structure

As addition of osmolytes can lead to protein folding, (21) we added 20% deuterated betaine to R67 DHFR to determine if osmolytes can provide order to the N-termini. SANS data were analyzed using GNOM; Figure 4A shows the pairwise distribution plot. The R_g was 22.84 ± 0.31 Å, which is larger than the value for R67 DHFR in the absence of betaine (e.g., 21.89 ± 0.12 Å). These results indicate a more swollen state in the presence of betaine. The ratio of I(0), scaled by sample concentration, for R67 DHFR in the presence of betaine to that in the absence of betaine was taken to ensure that the protein in 20% deuterated betaine was not aggregating. A ratio near 1 indicated that R67 DHFR in the presence of deuterated betaine was not aggregating under our experimental conditions.

Figure 4. SASSIE analysis of apo R67 DHFR in the presence of 20% deuterated betaine indicates compact as well as partially extended N-terminal conformations. (A) GNOM fits for the pairwise distribution of R_g for apo R67 DHFR with and without 20% deuterated betaine. SANS data were collected for R67 DHFR at 6.5 mg/mL in 20 mM deuterated Tris buffer in D₂O (pD 7.0) with 20% deuterated betaine (dashed purple line). A wider distribution in the presence of betaine and an R_g value of 23.0 ± 0.30 Å indicate an increased number of slightly more extended conformations for the termini of R67 DHFR. (B) Overlay of the density plots for the best frames with χ² values of <10 for the deuterated betaine protein sample (purple mesh) and for the apoprotein without betaine (green mesh).

SASSIE analysis of this SANS data set was performed using the same set of 461000 conformers that were used for the apoprotein. The χ² versus R_g plot (Figure S8A) also shows a “U” shape, indicating intermediate rather than very compact or very extended states fit the data well. Of all the structures generated using MD and Monte Carlo sampling, 58277 frames fit to the experimental SANS data with χ² values that are <10. The lowest χ² value was 3. Figure S8A shows a χ² = 10 cutoff for the good fits. The number of structures that fit the SANS data well has greatly increased (compared to that of apo R67 in buffer), again suggesting a more inflated structure. An overlay of the theoretical SANS profiles for the best and worst fits and the corresponding structures for these fits are shown in panels B and C of Figure S8, respectively. The R_g for the best structure is 23.79 Å, while that for the worst is 29.58 Å. The density plot (see Figure S8D) for the good fits (purple mesh) seems to occupy most of the space sampled by our set of 461000 frames (dark gray mesh). Also, the range of R_g values obtained indicates no (or transient) sampling of fully extended conformations for all four N-termini, which would have resulted in higher R_g values. The highest R_g sampled by Monte Carlo simulation is 29.49 Å, while our model of R67 DHFR with four fully extended N-termini has an R_g of 36.25 Å.

The R_g values for the good fits obtained using SASSIE ranged from 21.04 to 25.94 Å with an average R_g value of 22.78 ± 0.87 Å. The wider sampling range and higher average R_g both are consistent with the GNOM analysis, indicating that the N-termini sample extensive conformations in the presence of betaine. The COM point for Met1 in the four N-termini (see Figure S8E) indicates the termini sample many positions both near the core of the protein and farther from the surface. From these data and analyses, the disorder in the N-termini becomes more pronounced upon addition of betaine with the N-termini potentially acting as entropic bristles, sweeping out volume around the protein core. (44)

Osmolytes Probe Preferential Hydration of R67 DHFR

Hydration is important in the protein structure and function relationship. Three regions with different scattering-length densities are present in this experiment: (1) the protein, (2) the bulk solution of deuterated buffer containing hydrogenated osmolytes, and (3) the hydration shell surrounding the protein. Betaine or DMSO was used to probe the hydration of full-length R67 DHFR, while only betaine was used for our truncated R67 DHFR experiments. While a plot of R_g values obtained by GNOM analysis does not show any significant trend upon addition of an osmolyte (Figure S9), the zero-angle scattering intensity, I(0), is sensitive to changes in hydration. No significant change in the R_g value is consistent with the N-termini remaining disordered upon addition of an osmolyte. Note that, earlier, the R_g value of 22.8 ± 0.3 Å was obtained from the SANS data for apo R67 DHFR in 20% deuterated betaine. As deuterated betaine was added to the deuterated buffer, the contrast between the hydration layer and bulk was masked and the R_g value represents the overall shape of the protein without any contributions from the hydration layer. With hydrogenated betaine, the R_g is dependent upon the volume of the hydration layer and the location of the waters in the hydration shell. Even though the hydration layer contrast will increase with the addition of osmolytes, thus causing an apparent decrease in the overall R_g, our deuterated betaine data indicate the intrinsic protein volume increases. Therefore, the expansion of the intrinsic volume along with the apparent decrease in size from hydration contrast may be compensating for each other, and the net result is a relatively consistent R_g for R67 DHFR in the presence of hydrogenated osmolytes.

Decreasing I(0) values for both full-length and truncated R67 DHFR were observed with increasing concentrations of osmolytes. As shown in Figure 5, the data were fit to eq 2. The fits yield the volume of the hydration layer for R67 DHFR in the presence of osmolytes. The number of water molecules in the hydration layer is determined by dividing the observed water volume by the volume of a single water molecule (30 Å³). The number of osmolyte-excluding water molecules associated with the full-length protein is 1285 ± 214 or 1253 ± 199 using betaine or DMSO, respectively, indicating similar numbers of water molecules. The number of water molecules (n_w) excluding betaine from the hydration shell of the truncated protein is 380 ± 100. The difference in the number of hydrating waters (∼900) between full-length and truncated R67 DHFR indicates that the disordered N-terminal tails span a large volume in solution and are extensively hydrated.

Figure 5. Preferential hydration of the full-length and truncated R67 DHFR in the presence of osmolytes. Small angle neutron scattering intensity ratios with and without osmolyte [I(0)_s/I(0)] as a function of osmolyte concentration, f_v, for betaine (w/v) (magenta filled squares) and DMSO (v/v) (green filled triangles). Panels A and B show the data for full-length and truncated R67 DHFRs, respectively. Solid lines are fits to eq 2 to calculate the number of protein-associated waters, n_w.

To compare the experimental hydration values with theoretical numbers, the solvent accessible surface area (ASA) of tetrameric, truncated apo R67 DHFR (2RH2) was calculated to be 11072 Å² using the Molecular Operating Environment program (MOE 2015 version). If we assume the area of a water molecule to be 9 Å², (45) this yields approximately 1230 water molecules hydrating the truncated protein. For another high-resolution crystal structure of truncated R67 DHFR (2GQV), (9) the solvent accessible surface area was 11673 Å². This structure predicts ∼1297 water molecules.

To obtain a theoretical n_w value associated with the full-length R67 DHFR, we used the 7936 good fits obtained from our SASSIE analysis. The ASA was determined using SurfaceRacer (46) for each of the 7936 frames to obtain the n_w values. An average of 1800 water molecules were predicted in the hydration layer. Table 2 compares our experimental results with the predicted values from the truncated crystal structures as well as the average value for the 7936 full-length models of R67 DHFR. The predicted ranges of values for waters in the hydration shell are higher than those measured by our SANS data.

Table 2. Comparison of the Predicted and Experimental Numbers of Water Molecules (n_w) Hydrating R67 DHFR from the Crystal Structure, SASSIE Fits, and SANS Data

protein	source	predicted/experimental	no. of water molecules in the hydration layer (n_w)
truncated R67 DHFR	crystal structure (2RH2) (8)	predicted from ASA	1230^a
truncated R67 DHFR	crystal structure (2GQV) (9)	predicted from ASA	1297^b
truncated R67 DHFR	crystal structure (2GQV) (9)	experimental	340 in pore and first hydration shell
truncated R67 DHFR in the presence of betaine	SANS	experimental	380 ± 105
full-length R67 DHFR	frames from SASSIE analysis (χ² < 10)	predicted from ASA averaged for 7936 frames	1800
full-length R67 DHFR in the presence of betaine	SANS	experimental	1285 ± 214
full-length R67 DHFR in the presence of DMSO	SANS	experimental	1253 ± 199

^{^a}

The 2RH2 structure lacks 20 residues at the N-termini.

^{^b}

The 2GQV structure lacks 19 residues at the N-termini. Serine 20 was removed for comparison with 2RH2.

Effect of Osmolytes on the Thermal Stability of R67 DHFR

DSC scans were performed to monitor the effects of betaine and DMSO on the thermal stability of R67 DHFR. This is an additional way to determine if osmolytes are excluded from the protein surface. Previous thermal denaturation studies of R67 DHFR at pH 8 have found reversible folding with a melting temperature of 70.95 °C and evidence of an intermediate state. (47) Our DSC scans are shown in Figure 6. The data were fit to a three-state model, giving two melting temperatures (T_M) that correspond to two events in the thermal unfolding of R67 DHFR. T_M1 and T_M2 values in the absence of an osmolyte are 66.8 and 68.7 °C, respectively. Addition of 20% betaine increased the melting temperature of R67 DHFR by 2–3 °C, while 15% DMSO decreased the T_M by 7–9 °C (see Table S2). Stabilization of R67 DHFR in the presence of betaine is consistent with preferential exclusion of betaine from the protein surface. DMSO slightly destabilizes R67 DHFR, which indicates the likely interaction of DMSO with R67 DHFR, in the native or unfolded state.

Figure 6. (A) Effects of osmolytes on thermal denaturation of full-length R67 DHFR. DSC scans were performed with 150–160 μM R67 DHFR in MTA buffer with and without 20% betaine (magenta) and 15% DMSO (green). Betaine increases the melting temperature of the protein by 2–3 °C, whereas DMSO decreases it by 7–9 °C. (B) DSC thermograms for truncated R67 (50–90 μM) DHFR in MTA buffer, pH 8.0 buffer (black line), or buffer with 20% betaine (red) or 15% DMSO (cyan). Panels C and D show our PPC results. Thermal expansion coefficients (α_s) were obtained from PPC using 2.5 °C increments between 10 and 95 °C for (C) full-length R67 DHFR (90–150 μM) or (D) truncated R67 DHFR (99–150 μM). Experiments were performed in 45 mM Na₂HPO₄, pH 8.0 buffer alone (●), buffer with 10% betaine (○), or buffer with 20% betaine (△).

DSC was also performed on truncated R67 DHFR. The two T_M values were decreased 5 and 7 °C, respectively, compared to those of full-length protein, consistent with the disordered N-termini stabilizing the enzyme (see Table S2). Addition of 20% betaine to truncated R67 DHFR resulted in stabilization of both T_M values by 4 °C, while addition of 15% DSMO destabilized the protein by 7 °C. It has been reported that DSC of intrinsically disordered proteins or regions does not show an unfolding transition. (48,49) If true, then DSC signals of full-length and truncated R67 DHFR should report on the unfolding of the core “doughnut” structure. Also, addition of solutes should have similar effects on full-length and truncated R67 DHFRs. This behavior is seen in Figure 6. As addition of betaine increases the T_M values for both R67 DHFR species by similar levels (Table S2), it stabilizes the protein core, most likely by being excluded. In other words, the protein favors interaction with water. (22,50) In contrast, DMSO destabilizes the R67 core structure as the T_M values are lowered to similar degrees for both full-length and truncated protein species. This behavior suggests a preferential interaction mechanism for DMSO with the protein. (22,50) Again, DSC appears to report on the core, folded structure.

Pressure Perturbation Calorimetry

Another avenue for exploring the molar volume of R67 DHFR in the presence of betaine uses pressure perturbation calorimetry. From PPC, the thermal expansion coefficient for R67 DHFR was 8.7 × 10^–4 K^–1 at 10 °C (Table 3), and it decreased as the temperature increased to 57 °C (Figure 6C). Structure-breaking polar groups on the surface of the protein are most likely responsible for the decrease in α_s. (38) The denaturation transition of R67 DHFR between 60 and 75 °C caused an increase in α_s, which indicates an increase in the volume of R67 DHFR as the protein denatures. Integrating the area underneath this peak in the thermogram yielded a relative change in volume (ΔV/V) of +0.0013. A T_M for the denaturation of R67 DHFR of 67.5 °C was calculated from the PPC. This value matches well with the conventional DSC analysis of R67 DHFR at pH 8 (Table S2). (47)

Table 3. Analysis of the PPC Data for Full-Length and Truncated R67 DHFRs in 45 mM Na₂HPO₄, pH 8.0 Buffer with 0, 10, or 20% (w/w) Betaine

DHFR	[betaine] (%)	ΔV/V	α_s10–40 (×10^–4 K^–1)	T_M (°C)	α_s10 (×10^–4 K^–1)	Δα_s (×10^–5 K^–1)
full-length R67 DHFR	0	0.0013	2.7	67.5	8.7	4.3
	10	0.00077	3.1	70.0	9.7	4.8
	20	0.00048	3.2	72.4	8.9	3.3
truncated R67 DHFR	0	0	9.7	nd^a	22.1	nd^a
	10	0	8.2	nd^a	19.3	nd^a
	20	0	5.1	nd^a	14.5	nd^a

^{^a}

Not determined as there was no thermal denaturation transition in the PPC thermogram.

As a control, PPC was also performed on the truncated form of the protein (see Figure 6D). The α_s at 10 °C for truncated R67 DHFR (2.2 × 10^–3 K^–1) was twice that of the full-length protein (Table 3). Another interesting characteristic of the PPC for the truncated R67 DHFR was that no transition for denaturation was noted (Figure 6D). As there is a clear transition in the DSC data for truncated DHFR (see Figure 6B), the lack of a denaturation transition in the PPC thermogram is not due to the protein being unfolded. Additionally, we note the truncated enzyme was active, indicating that it was not unfolded.

A balance exists between elements that contribute to the negative volume change (i.e., loss of voids in the protein and the electrostriction of water around polar and charged groups that are more exposed upon unfolding) and those that contribute to a positive volume change (a larger thermal expansivity for the unfolded state vs the folded state and changes in the hydrophilic–hydrophobic balance of the exposed groups). (38,51) Additional effects may be loss of clathrate water in the R67 active site pore (9) and dissociation of a tetramer to four unfolded monomers. The relative contribution of these effects leads to the observed α value. For a positive ΔV/V (as in full-length R67 DHFR), the positive effects must predominate. For ΔV/V to be zero (as in truncated R67 DHFR), the various effects appear to be balanced. Most monomeric, globular proteins show negative ΔV/V values. (51,52) Because the structural differences between the truncated and full-length R67 DHFRs are the four disordered N-termini, they appear to be the key determinant for the positive ΔV/V value seen for full-length R67.

PPC thermograms were also performed in the presence of betaine for both full-length and truncated R67 DHFRs (Figure 6). Addition of 10% (w/w) betaine to the full-length protein caused an increase in the α_s value at 10 °C (9.7 × 10^–4 K^–1) relative to the protein in the absence of betaine (Table 3). This increase in α_s suggests there is an increase in the protein volume that is likely due to extension of the collapsed N-termini in the presence of betaine. Further increasing the betaine concentration to 20% (w/w) decreased the α_s at 10 °C to 8.9 × 10^–4 K^–1, similar to the value in the absence of betaine. This reduction most likely describes a decrease in the size of the solvation shell of the full-length protein. Similar effects of various solutes on the α values for RNase (38) and SNase (53) have been previously observed and ascribed to effects on the hydration shell. The increased volume at 10% betaine correlates with our SANS result of an increased R_g for full-length R67 DHFR in buffer containing 20% deuterated betaine.

For truncated R67 DHFR, the α_s value at 10 °C decreases with each increase in betaine concentration from 1.9 × 10^–3 K^–1 at 10% betaine to 1.5 × 10^–3 K^–1 at 20% betaine. The volume of the truncated protein, including its water shell, decreases as betaine is added, decreasing the concentration of water in the solution.

Discussion

Click to copy section linkSection link copied!

The R67 DHFR monomer is 78 amino acids long, and around 16–20 N-terminal residues are disordered; therefore, ∼20–25% of its sequence is unstructured. R67 assumes a compact structure by forming a homotetramer. Chymotrypsin treatment of the folded protein results in a truncated product, which is almost fully active but 2.6 kcal/mol less stable. (7) Expression of the truncated protein from a shorter gene sequence does not confer trimethoprim resistance. Thus, the N-termini are essential for protein expression and/or stability but not for catalysis. To understand the conformational space sampled by the N-termini of R67 DHFR, we characterized full-length and truncated R67 DHFR using SANS.

Apoprotein Analysis

The best fits for apo R67 DHFR indicate compaction of two N-termini on one side of the ordered tetramer core, whereas the other two N-termini prefer to remain partially extended (see Figure 2D and Figure S5). In many of these poses, the N-terminal residues interact with each other and/or with residues exposed on the monomer–monomer interface. Intramolecular and intermolecular interactions are both feasible. These interactions lead to compaction of the overall shape and seem likely to be why the N-termini provide 2.6 kcal/mol of stability to R67 DHFR. (7)

Data mining of the conformers fitting the SANS profile was accomplished using a Python script. Frequent interactions were identified by counting the number of times the COM of each amino acid occurs within 5 Å of the COM of every other residue. Figure S10A shows a heat map of the minimum distance between residues. Figure S10B provides a heat map of the number of these interactions versus the amino acid number (1–78 for the first monomer, 79–156 for the second monomer, 157–234 for the third monomer, and 235–312 for the last monomer). The symmetry of the structure provides an initial understanding of these plots as monomers nearby in space interact (A and C or B and D), while distant monomers do not. In Figure S10, intramolecular interactions can be visualized by the points near the diagonal while intermolecular interactions are indicated by the areas describing interactions between residues 1–78 and 157–234 (for example).

Using the symmetry of the core structure, the number of potential interactions was summed, using the rationale that a stabilizing interaction would occur in more than one monomer. Supplemental Excel sheet 1 lists these amino acid pairs. Three bins were noted: first, pairs that occur more than 1000 times (=22); second, intramolecular pairs that occur in all four monomers (=8); and third, intermolecular pairs that occur in all four monomers (=1). Hydrophobic, polar/uncharged, and charged residues are identified and colored in the excel sheet as described by Eisenberg et al. (54) In the pairs that occur >1000 times, hydrophobic residues occur 50% of the time while polar/uncharged amino acids occur 40% of the time and charged residues 10% of the time. These pairs mostly describe N-terminal to N-terminal interactions.

As the N-terminal sequence contains several hydrophobic side chains (M1, I2, V8, A13, F16, V17, and F18), these amino acids could also potentially form hydrophobic interactions with similar exposed side chains on the folded protein surface. In particular, each of the two-symmetry related W45 residues provides ∼94 Å² of ASA for interaction. Short distances were observed from most of the hydrophobic residues mentioned above to W45 and its symmetry-related W201 residue. Also, cation−π interactions could be transiently occurring as R3 often occurred nearby W45 as well as M1 (N-terminal residue).

Other residues contributing to hydrophobic surfaces near the monomer–monomer interface include A22, F24, M26, V30, V43, V71, A72, and I77. Spatial proximity was noted for several N-terminal residues with A22, M26, and V43. Interestingly, three 2-methylpentane-2,4-diol molecules per monomer were found close to the A22, W45, and L50 residues in the 2RH2 structure, (8) suggesting a possible hydrophobic interaction hot spot. Figure 7 shows exposed hydrophobic residues on the ASA surface of R67 DHFR.

Figure 7. Surface of R67 DHFR (2RH2) shown with the exposed lipophilic surfaces colored green and hydrophilic surfaces colored magenta along the monomer–monomer interface. The surface was made partially transparent so the side chains could be seen. Two symmetry-related W45 residues are labeled in the center of the figure. This figure was composed using the lipophilic surface option in MOE (version 2015.10).

The crystal structure of truncated R67 DHFR shows exact 222 symmetry. (4) While this symmetry could also apply to each of the disordered N-termini, it is more likely that they impart asymmetry via their disorder.

Analysis of Binary and Ternary Complexes

Table 2 summarizes the various R_g values obtained from GNOM and SASSIE analysis. No substantial effect of ligand binding was observed in the conformations sampled by the N-termini as those frames that provided the best fits to the SANS data for both the binary and ternary complexes mostly overlap with those sampled by the apoprotein. GNOM analysis yielded comparable R_g values for the apoprotein and ligand-bound protein samples, and the conformers obtained from our SASSIE analysis placed the disordered tails near the sides of the active site pore.

Data mining of the ternary complex conformers that fit the SANS data was also performed. Figure S11A plots the minimum distance between the COM of residues. A pattern similar to that seen in apo R67 DHFR is observed. Supplementary Excel sheet 2 lists those amino acid pairs whose centers of mass are ≤5 Å apart. Three bins were again considered: 47 pairs occur more than 1000 times, while nine intramolecular and two intermolecular pairs occur in all four monomers. The same type of interactions are observed as in the apo conformers with hydrophobic residues occurring 56% of the time, polar/uncharged 32% of the time, and charged 13% of the time. One difference is that the N-terminal methionines now very frequently interact with W45 or W201 (symmetry-related residues).

Effects of Osmolytes

The main difference in our data arises when betaine is added, which leads to a more swollen state of R67 DHFR. Osmolytes that are excluded from protein surfaces are known to stabilize the protein via the preferential exclusion mechanism. (22) The ability of TMAO to force folding of a modified RNase was attributed to its preferential exclusion from the peptide backbone (also termed the solvophobic effect). (21,55) While R67 DHFR was found to be stabilized upon addition of betaine by our DSC studies, no disorder to order transition was observed for the disordered tails from our analysis of the SANS data. On the contrary, SASSIE finds the addition of betaine results in greater conformational sampling of the disordered tails, from being collapsed near the sides of the protein to being partially extended. In our previous studies of the interaction of betaine with folate and other compounds, we found betaine can compete with water to form stable interactions. (56) Betaine prefers to interact with aromatic surfaces as well as cationic and amide nitrogen atoms, while water prefers to interact with carboxylate, phosphate amide and hydroxyl oxygens. (56,57) For the case of R67 DHFR, betaine may interact with some residues in the N-termini, hindering the collapsed conformations from being sampled.

Another possible explanation for the extensive sampling of the disordered tails upon addition of betaine may be attributed to changes in the solvent structure. Studies have characterized the effects of solutes on the structure of bulk as well as hydrating water molecules around proteins. (37,38) The nature and extent of these alterations depend on the chemical properties of the solutes. Polar and hydrophilic surfaces were found to be water structure breakers, whereas hydrophobic surfaces were described as water structure makers. (37) Stabilization of RNase A by 1.5 M sucrose was previously observed, while accompanying pressure perturbation calorimetry studies showed nonlinear effects on α, the apparent coefficient of thermal expansion, Specifically, RNase is less compact in 0.5 M sucrose, as indicated by an increased α, than in the absence of sucrose, while the protein becomes more compact at 1.5 M sucrose, yielding a decreased α. (38) The differences in α were attributed to changes in protein hydration.

Hydration Studies

Experiments that have examined protein hydration have used varying techniques. A typical approach calculates the accessible surface area (ASA) and divides the value by 9 Å² to predict the number of solvent waters in the hydration shell. This yields a high value. In contrast, experimental approaches often yield smaller numbers of hydration waters. For lysozyme, ASA calculations predict ∼900 waters of hydration. (27) Experimental techniques for studying lysozyme hydration include NMR, (58) excess heat capacity, (59) dielectric relaxation, (60) and X-ray diffraction. (61) These experimental approaches yield 121–900 hydration waters, indicating the value is sensitive to the technique used as well as the experimental conditions employed. A previous SANS study of hydration in lysozyme used different osmolytes. (27) With added betaine, triethylene glycol, PEG400, or PEG1000, 84 ± 5, 114 ± 24, 156 ± 8, or 347 ± 11 hydration waters were observed, respectively, along with different water shell thicknesses. The increase in the number of waters (n_w) may be due to osmotic stress effects combined with volume exclusion as the size of the osmolyte increases. (45,62,63) Alternatively, fewer waters may be observed if the osmolyte interacts with the protein surface. Both factors likely play a role in observation of an n_w value that is lower than the predicted upper limit.

In our SANS studies of R67 DHFR, we used the osmoprotectant, betaine, as it is often excluded from the protein surface. (64) Our SANS experiments allow three areas of different contrast to be delineated: the protein, the bulk solution containing osmolytes, and the hydration shell that excludes osmolytes. The number of water molecules (n_w) responsible for the exclusion of betaine from the truncated R67 DHFR surface was found to be 380 ± 105. This value is smaller than the predicted value of 1230–1297 waters from ASA calculations from the crystal structures (2RH2 (8) and 2GQV (9)).

Because of the high resolution and low temperature factors of the 1.1 Å resolution structure of R67 DHFR (2GQV), 85 waters per monomer were identified in the first hydration shell (e.g., formation of a H-bond with the protein surface) and 106 in higher-level shells. (9) This yields 340 waters in the first hydration shell of the tetramer. This value compares to that from our SANS experiment with truncated R67 DHFR that yields an n_w value of 380 ± 100. Thus, both SANS and crystallography appear to measure polar bound waters.

When SANS was performed on full-length R67 DHFR, addition of both betaine and DMSO yielded n_w values of ∼1250 waters hydrating the protein surface. This value is lower than the average n_w value of 1800 waters predicted using ASA calculations on PDB files generated by MD and directed Monte Carlo analyses in SASSIE. We also counted the average number of waters associated with the R67 DHFR tetramer in our MD trajectories and found an average of 1647 (range of 1446–1879). Again, the experimental value is lower than the predicted upper limit, suggesting some level of interaction of the osmolyte with the protein surface.

When the n_w values for truncated (380) and full-length R67 DHFRs (1200) are compared, the difference is 900 waters. This indicates each N-terminus is well-hydrated by ∼225 waters.

To test whether betaine and DMSO were interacting with R67 DHFR, we performed DSC experiments. Excluded osmolytes typically increase the stability of proteins by increasing the level of hydration, while interacting osmolytes decrease protein stability. (21,65) Additionally, DSC experiments of intrinsically disordered proteins (IDPs) typically lack cooperative structural transitions, (48,49,66) so our results appear to report on the effects of the osmolyte on the structural core of the protein. This idea is supported by a similar 4–5 °C increase in T_M when betaine is added to either truncated or full-length R67 DHFR. Thus, betaine appears to be mostly excluded from the surface of the core of the R67 DHFR structure (supported by DSC results), while there is some level of interaction of the osmolyte with the disordered N-termini (supported by our deuterated betaine SANS and PPC results).

Addition of 20% betaine increased the T_M values by 4–5 °C for both full-length and truncated R67 DHFRs, while addition of 20% DMSO decreased the T_M values by 5–7 °C. These results were surprising given that the numbers of hydrating waters for these two osmolytes were within error as measured by our SANS experiments. Though the n_w values are similar, the water location may vary. DMSO can form hydrophobic interactions, whereas betaine interacts with aromatic, amide, and cationic nitrogens exposed on the protein. Thus, both osmolytes may lead to the exclusion of water from different protein surfaces, which can in turn result in the variable effects on protein stability.

Conclusion

Click to copy section linkSection link copied!

While it is confounding that disordered regions can provide some level of stability to a folded protein, we find this is the case for R67 DHFR. From our SANS data, we find the disordered N-termini prefer to sample conformational space near the sides of the apoprotein. This allows both N-termini to interact with themselves as well as the monomer–monomer interface, providing 2.6 kcal/mol of stability to R67 DHFR. (7)

According to van der Lee et al., (67) entropic chains are a form of IDP that remain disordered. This applies to the N-termini of R67 DHFR as they do not fold upon addition of a ligand. Addition of betaine to R67 DHFR results in a larger R_g and SASSIE fits that predict a wider sampling volume. These results suggest the N-termini are responsive to their environment. It is tempting to speculate that in the cell, the N-termini may interact in a similar fashion with small molecules or macromolecules and provide an entropic bristle function where they sweep out volume around the protein core. This would prevent large molecules from entering this space but allow penetration of small molecules. (68) Entropic bristles have also been proposed to enhance protein solubility and prevent aggregation. (44,69−71) Finally, we note there are several R-plasmid DHFRs that differ only in the sequence of their N-termini. (10−12) All have N-termini of similar lengths, which indicates it may be the length of the N-termini more than the sequence that is important for its function. A longer disordered sequence (as in our His-tag constructs cloned in pRSETB with an additional 30 amino acids) leads to ∼2-fold increases in K_m values for NADPH and DHF. (72) Another study found the R67 DHFR N-terminal sequence was essential for evolvability. (73) These various observations suggest that R67 DHFR’s disordered N-termini play roles in stability, solubility, evolvability, and substrate access.

Finally, both the betaine studies and the preferential hydration measurements indicate the disordered tails are highly hydrated, consistent with large, exposed surface areas. These data sets support the importance of water and solutes in the R67 structure–function relationship. As the disordered segments are exposed and our SANS results show the polar regions are well hydrated, it seems likely that betaine can compete well with water for solvation of aromatic groups. Indeed, Uversky suggested intrinsically disordered proteins (IDPs) are “multifarious interactors”. (68) While he meant IDP can often interact with various protein partners, here we wonder if disordered regions can interact with different solutes, which can subtly change their behavior. This would add another layer of complexity to the role of IDP and disordered regions in the cell.

Supporting Information

Click to copy section linkSection link copied!

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00822.

A 20% SDS–PAGE gel indicating clear separation of full-length and truncated R67 DHFRs, a flow diagram of our steps using MD and SASSIE to find N-terminal conformers that fit the SANS data, another figure depicting the SANS profile and GNOM analysis for truncated R67 DHFR as well as a dimensionless Kratky plot comparing full-length and truncated R67, the COM position for the N-terminal methionine of apo R67 DHFR conformers that fit the SANS data, SASSIE analysis of the data of the binary complex (R67 DHFR–NADP⁺), the ternary complex (R67 DHFR–NADP⁺–DHF), and R67 DHFR in the presence of 20% deuterated betaine, plots of R_g for full-length and truncated R67 DHFR probed after addition of betaine or DMSO, data mining plots for apo and ternary complex fits, and two tables listing R_g values from the various programs and T_M values from DSC data (PDF)
Excel sheet from the data mining of apo conformers (XLSX)
Excel sheet from the data mining of ternary conformers (XLSX)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

Click to copy section linkSection link copied!

Corresponding Author
- Elizabeth E. Howell - Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, United States; Genome Science and Technology Program, University of Tennessee, Knoxville, Tennessee 37996-0840, United States; http://orcid.org/0000-0001-6157-433X; Email: [email protected]
Authors
- Purva P. Bhojane - Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, United States
- Michael R. Duff Jr. - Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, United States
- Khushboo Bafna - Genome Science and Technology Program, University of Tennessee, Knoxville, Tennessee 37996-0840, United States
- Pratul Agarwal - Computer Science and Mathematics Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States; http://orcid.org/0000-0002-3848-9492
- Christopher Stanley - Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States; http://orcid.org/0000-0002-4226-7710
Funding
This work was supported by National Institutes of Health Grants GM 110669 (to E.E.H.) and GM105978 (to P.A.).
Notes
The authors declare no competing financial interest.

Acknowledgments

Click to copy section linkSection link copied!

A portion of this research at ORNL’s Spallation Neutron Source was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. The authors thank Susan Krueger from the National Institute of Standards and Technology (NIST) for her help with SASSIE analysis. This work benefitted from CCP-SAS software developed through a joint EPSRC (EP/K039121/1) and NSF (CHE-1265821) grant.

Abbreviations

Click to copy section linkSection link copied!

ASA	accessible surface area
COM	center of mass
DHF	dihydrofolate
DHFR	dihydrofolate reductase
DMSO	dimethyl sulfoxide
DSC	differential scanning calorimetry
IDP	intrinsically disordered protein
I(0)	zero-angle scattering intensity
MD	molecular dynamics
MTA buffer	100 mM MES, 50 mM Tris, and 50 mM acetic acid
NADP⁺ and NADPH	oxidized and reduced nicotinamide adenine dinucleotide phosphate, respectively
NMR	nuclear magnetic resonance
NTA	nitrilotriacetic acid
n_w	number of water molecules
PDB	Protein Data Bank
PEG	polyethylene glycol
PPC	pressure perturbation calorimetry
q	scattering angle
R_g	radius of gyration
SANS	small angle neutron scattering
THF	tetrahydrofolate
T_M	melting temperature.

References

Click to copy section linkSection link copied!

This article references 73 other publications.

1
Toulouse, J. L., Edens, T. J., Alejaldre, L., Manges, A. R., and Pelletier, J. N. (2017) Integron-Associated DfrB4, a Previously Uncharacterized Member of the Trimethoprim-Resistant Dihydrofolate Reductase B Family, Is a Clinically Identified Emergent Source of Antibiotic Resistance. Antimicrob. Agents Chemother. 61, e02665-16, DOI: 10.1128/AAC.02665-16
Google Scholar
There is no corresponding record for this reference.
2
Bastien, D., Ebert, M. C., Forge, D., Toulouse, J., Kadnikova, N., Perron, F., Mayence, A., Huang, T. L., Vanden Eynde, J. J., and Pelletier, J. N. (2012) Fragment-based design of symmetrical bis-benzimidazoles as selective inhibitors of the trimethoprim-resistant, type II R67 dihydrofolate reductase. J. Med. Chem. 55, 3182– 3192, DOI: 10.1021/jm201645r
Google Scholar
2
Fragment-Based Design of Symmetrical Bis-benzimidazoles as Selective Inhibitors of the Trimethoprim-Resistant, Type II R67 Dihydrofolate Reductase
Bastien, Dominic; Ebert, Maximilian C. C. J. C.; Forge, Delphine; Toulouse, Jacynthe; Kadnikova, Natalia; Perron, Florent; Mayence, Annie; Huang, Tien L.; Vanden Eynde, Jean Jacques; Pelletier, Joelle N.
Journal of Medicinal Chemistry (2012), 55 (7), 3182-3192CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
The continuously increasing use of trimethoprim as a common antibiotic for medical use and for prophylactic application in terrestrial and aquatic animal farming has increased its prevalence in the environment. This has been accompanied by increased drug resistance, generally in the form of alterations in the drug target, dihydrofolate reductase (DHFR). The most highly resistant variants of DHFR are known as type II DHFR, among which R67 DHFR is the most broadly studied variant. We report the first attempt at designing specific inhibitors to this emerging drug target by fragment-based design. The detection of inhibition in R67 DHFR was accompanied by parallel monitoring of the human DHFR, as an assessment of compd. selectivity. By those means, small arom. mols. of 150-250 g/mol (fragments) inhibiting R67 DHFR selectively in the low millimolar range were identified. More complex, sym. bis-benzimidazoles and a bis-carboxyphenyl were then assayed as fragment-based leads, which procured selective inhibition of the target in the low micromolar range (Ki = 2-4 μM). The putative mode of inhibition is discussed according to mol. modeling supported by in vitro tests.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XktVynsLo%253D&md5=e648c46e90ea8930a85a0b4886b468c2
3
Toulouse, J. L., Abraham, S. M. J., Kadnikova, N., Bastien, D., Gauchot, V., Schmitzer, A. R., and Pelletier, J. N. (2017) Investigation of Classical Organic and Ionic Liquid Cosolvents for Early-Stage Screening in Fragment-Based Inhibitor Design with Unrelated Bacterial and Human Dihydrofolate Reductases. Assay Drug Dev. Technol. 15, 141– 153, DOI: 10.1089/adt.2016.768
Google Scholar
3
Investigation of Classical Organic and Ionic Liquid Cosolvents for Early-Stage Screening in Fragment-Based Inhibitor Design with Unrelated Bacterial and Human Dihydrofolate Reductases
Toulouse, Jacynthe L.; Abraham, Sarah M. J.; Kadnikova, Natalia; Bastien, Dominic; Gauchot, Vincent; Schmitzer, Andreea R.; Pelletier, Joelle N.
Assay and Drug Development Technologies (2017), 15 (4), 141-153CODEN: ADDTAR; ISSN:1540-658X. (Mary Ann Liebert, Inc.)
Drug design by methods such as fragment screening requires effective solubilization of millimolar concns. of small org. compds. while maintaining the properties of the biol. target. We investigate four org. solvents and three 1-butyl-3-methylimidazolium (BMIm)-based ionic liqs. (ILs) as cosolvents to establish conditions for screening two structurally unrelated dihydrofolate reductases (DHFRs) that are prime drug targets. Moderate concns. (10%-15%) of cosolvents had little effect on inhibition of the microbial type II R67 DHFR and of human DHFR (hDHFR), while higher concns. of org. cosolvents generally decreased activity of both DHFRs. In contrast, a specific IL conserved the activity of one DHFR, while severely reducing the activity of the other, and vice versa, illustrating the differing effect of ILs on distinct protein folds. Most of the cosolvents investigated preserved the fold of R67 DHFR and had little effect on binding of the cofactor NADPH, but reduced the productive affinity for its substrate. In contrast, cosolvents resulted in modest structural destabilization of hDHFR with little effect on productive affinity. We conclude that the org. cosolvents, methanol, DMF, and dimethylsulfoxide, offer the most balanced conditions for early-stage compd. screening as they maintain sufficient biol. activity of both DHFRs while allowing for compd. dissoln. in the millimolar range. However, IL cosolvents showed poor capacity to solubilize org. compds. at millimolar concns., mitigating their utility in early-stage screening. Nonetheless, ILs could provide an alternative to classical org. cosolvents when low concns. of inhibitors are used, as when characterizing higher affinity inhibitors.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVantrfO&md5=0498186b3b64f5d33a9473b9c5bca87a
4
Narayana, N., Matthews, D. A., Howell, E. E., and Xuong, N. (1995) A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site,. Nat. Struct. Mol. Biol. 2, 1018– 1025, DOI: 10.1038/nsb1195-1018
Google Scholar
4
A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site
Narayana, Narendra; Matthews, David A.; Howell, Elizabeth E.; Xuong, Nguyen-huu
Nature Structural Biology (1995), 2 (11), 1018-25CODEN: NSBIEW; ISSN:1072-8368. (Nature Publishing Co.)
Bacteria expressing R67-plasmid encoded dihydrofolate reductase (R67 DHFR) exhibited high-level resistance to the antibiotic trimethoprim. Native R67 DHFR is a 34,000 Mr homotetramer which exists in equil. with an inactive dimeric form. The structure of native R67 DHFR has now been solved at 1.7 Å resoln. and is unrelated to that of chromosomal DHFR. Homotetrameric R67 DHFR has an unusual pore, 25 Å in length, passing through the middle of the mol. Two folate mols. bind asym. within the pore indicating that the enzyme's active site consists of symmetry related binding surfaces from all four identical units.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXptlegtrc%253D&md5=23ff4e76931aeef868529123d7f91a02
5
Feng, J., Grubbs, J., Dave, A., Goswami, S., Horner, C. G., and Howell, E. E. (2010) Radical redesign of a tandem array of four R67 dihydrofolate reductase genes yields a functional, folded protein possessing 45 substitutions. Biochemistry 49, 7384– 7392, DOI: 10.1021/bi1005943
Google Scholar
5
Radical Redesign of a Tandem Array of Four R67 Dihydrofolate Reductase Genes Yields a Functional, Folded Protein Possessing 45 Substitutions
Feng, Jian; Grubbs, Jordan; Dave, Ashita; Goswami, Sumit; Horner, Caroline Glyn; Howell, Elizabeth E.
Biochemistry (2010), 49 (34), 7384-7392CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
R67 dihydrofolate reductase (DHFR) is a plasmid-encoded, type II enzyme. Four monomers (78 amino acids long) assemble into a homotetramer possessing 222 symmetry. In previous studies, a tandem array of four R67 DHFR gene copies was fused in frame to generate a functional monomer named Quad1. This protein possessed the essential tertiary structure of the R67 "parent". To facilitate mutagenesis reactions, restriction enzyme sites were introduced in the tandem gene array. S59A and H362L mutations were also added to minimize possible folding topologies; this protein product, named Quad3, possesses 10 substitutions and is functional. Since R67 DHFR possesses a stable scaffold, a large jump in sequence space was performed by the further addn. of 45 amino acid substitutions. The mutational design utilized alternate sequences from other type II DHFRs. In addn., most of the mutations were positioned on the surface of the protein as well as in the disordered N-terminal sequence, which serves as the linker between the fused domains. The resulting Quad4 protein is quite functional; however, it is less stable than Quad1, suffering a ΔΔG loss of 5 kcal/mol at pH 5. One unexpected result was formation of Quad4 dimers and higher order oligomers at pH 8. R67 DHFR, and its deriv. Quad proteins, possesses a robust scaffold, capable of withstanding introduction of ≥55 substitutions.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXpslKjs7s%253D&md5=635f32ee7e52a6c1325ee7f76c8a65ca
6
Matthews, D. A., Smith, S. L., Baccanari, D. P., Burchall, J. J., Oatley, S. J., and Kraut, J. (1986) Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67. Biochemistry 25, 4194– 4204, DOI: 10.1021/bi00363a005
Google Scholar
6
Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67
Matthews, David A.; Smith, S. L.; Baccanari, D. P.; Burchall, J. J.; Oatley, S. J.; Kraut, J.
Biochemistry (1986), 25 (15), 4194-204CODEN: BICHAW; ISSN:0006-2960.
Cryst. R67 dihydrofolate reductase (DHFR) is a dimeric mol. with 2 identical 78-amino-acid-residue subunits, each folded into a β-barrel conformation. The outer surfaces of the 3 longest β-strands in each protomer together form a 3rd β-barrel having 6 strands at the subunit interface. A unique feature of the enzyme structure is that while the intersubunit β-barrel is quite regular over most of its surface, an 8-Å gap runs the full length of the barrel, disrupting potential H bonds between β-strand D in subunit I and the adjacent corresponding strand of subunit II. It is proposed that this deep groove is the NADPH-binding site and that the assocn. between protein and cofactor is modulated by H-bonding interactions along one face of this antiparallel β-barrel structure. A hypothetical model is proposed for the R67 DHFR-NADPH-folate ternary complex that is consistent with both the known reaction stereoselectivity and the weak binding of 2,4-diamino inhibitors to the plasmid-specified reductase. Geometrical comparison of this model with an exptl. detd. structure for chicken DHFR suggests that chromosomal and type II R-plasmid-specified enzymes may have independently evolved similar catalytic machinery for substrate redn.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XksFKqtrk%253D&md5=00f5fc4a2ebdeea18be9e89909f7d2c7
7
Reece, L. J., Nichols, R., Ogden, R. C., and Howell, E. E. (1991) Construction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein. Biochemistry 30, 10895– 10904, DOI: 10.1021/bi00109a013
Google Scholar
7
Construction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein
Reece, Lisa J.; Nichols, Robert; Ogden, Richard C.; Howell, Elizabeth E.
Biochemistry (1991), 30 (45), 10895-904CODEN: BICHAW; ISSN:0006-2960.
Plasmid R67 dihydrofolate reductase (DHFR) is a novel protein that provides clin. resistance to the antibacterial drug, trimethoprim. The previously published crystal structure of a dimeric form of R67 DHFR indicated that the 1st 16 amino acids are disordered. To investigate whether these amino acids are necessary for protein function, the 1st 16 N-terminal residues were cleaved off by chymotrypsin. The truncated protein was found to be fully active with kcat = 1.3 s-1, Km(NADPH) = 3.0 μM, and Km(dihydrofolate) = 5.8 μM. This result suggests that the functional core of the protein resides in the β-barrel structure defined by residues 27-78. To study this protein further, synthetic genes coding for full-length and truncated R67 DHFRs were constructed. Surprisingly, the gene coding for truncated R67 DHFR did not produce protein in vivo or confer trimethoprim resistance upon Escherichia coli. Therefore, the relative stabilities of native and truncated R67 DHFR were investigated by equil. unfolding studies. The unfolding of dimeric native R67 DHFR was protein concn.-dependent and could be described by a 2-state model involving native dimer and unfolded monomer. Using absorbance, fluorescence, and CD techniques, an av. ΔGH2O of 13.9 kcal/mol was found for native R67 DHFR. In contrast, an av.ΔGH2O of 11.3 kcal/mol was obsd. for truncated R67 DHFR. These results indicated that native R67 DHFR is 2.6 kcal/mol more stable than the truncated protein. This stability difference may be part of the reason why protein from the truncated gene is not found in vivo in E. coli.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXmslCntbc%253D&md5=1191aeea031ad497bff25437b10c4d57
8
Krahn, J. M., Jackson, M. R., DeRose, E. F., Howell, E. E., and London, R. E. (2007) Crystal structure of a type II dihydrofolate reductase catalytic ternary complex. Biochemistry 46, 14878– 14888, DOI: 10.1021/bi701532r
Google Scholar
8
Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex
Krahn, Joseph M.; Jackson, Michael R.; DeRose, Eugene F.; Howell, Elizabeth E.; London, Robert E.
Biochemistry (2007), 46 (51), 14878-14888CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a sym. homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR·DHF·NADP+ catalytic complex, resolved to 1.26 Å. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine ring. This set of interactions orients the arom. rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Addnl., a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67'-Y69' residues provides an unusually tight interface, which appears to serve as a "mol. clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addn. to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationally challenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlOnur3O&md5=68e1db370224ac27699b3a46cdf5c49c
9
Narayana, N. (2006) High-resolution structure of a plasmid-encoded dihydrofolate reductase: pentagonal network of water molecules in the D2-symmetric active site. Acta Crystallogr., Sect. D: Biol. Crystallogr. 62, 695– 706, DOI: 10.1107/S0907444906014764
Google Scholar
There is no corresponding record for this reference.
10
Amyes, S. G. and Smith, J. T. (1976) The purification and properties of the trimethoprim-resistant dihydrofolate reductase mediated by the R-factor, R388. Eur. J. Biochem. 61, 597– 603, DOI: 10.1111/j.1432-1033.1976.tb10055.x
Google Scholar
10
The purification and properties of the trimethoprim-resistant dihydrofolate reductase mediated by the R-factor, R388
Amyes, Sebastian G. B.; Smith, John T.
European Journal of Biochemistry (1976), 61 (2), 597-603CODEN: EJBCAI; ISSN:0014-2956.
The R-factor, R 388, mediates the prodn. of a trimethoprim-resistant dihydrofolate reductase. This enzyme has a different mol. wt. and pH profile than the trimethoprim-sensitive enzyme of the Escherichia coli host. The R-factor mediated enzyme was sepd. completely from the host E. coli enzyme by DEAE-cellulose ion-exchange chromatog. The purified R-factor enzyme was ∼20,000 times less susceptible to trimethoprim than the E. coli enzyme and although it was inhibited competitively by trimethoprim, its Ki was 20,000 times greater than that of the host enzyme. The R388 and E. coli enzymes also differed in their substrate specificity requirements. In addn., the R388 enzyme surprisingly conferred high level resistance to the broad spectrum dihydrofolate reductase inhibitor, amethopterin. The possible origins of the R388 enzyme are discussed.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XhtVWgtr0%253D&md5=431d7961c3c4d8ebf230b6f2d31c66ae
11
Flensburg, J. and Steen, R. (1986) Nucleotide sequence analysis of the trimethoprim resistant dihydrofolate reductase encoded by R plasmid R751. Nucleic Acids Res. 14, 5933, DOI: 10.1093/nar/14.14.5933
Google Scholar
There is no corresponding record for this reference.
12
Alonso, H. and Gready, J. E. (2006) Integron-sequestered dihydrofolate reductase: a recently redeployed enzyme. Trends Microbiol. 14, 236– 242, DOI: 10.1016/j.tim.2006.03.003
Google Scholar
12
Integron-sequestered dihydrofolate reductase: A recently redeployed enzyme
Alonso, Hernan; Gready, Jill E.
Trends in Microbiology (2006), 14 (5), 236-242CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)
A review. The introduction and wide use of antibacterial drugs has resulted in the emergence of resistant organisms. DfrB dihydrofolate reductase (DHFR) is a bacterial enzyme that is uniquely assocd. with mobile gene cassettes within integrons, and confers resistance to the drug trimethoprim. This enzyme has intrigued microbiologists since it was discovered more than thirty years ago because of its simple structure, enzymic inefficiency and its virtual insensitivity to trimethoprim. Here, for the first time, a comprehensive discussion of genetic, evolutionary, structural and functional studies of this enzyme is presented together. This information supports the ideas that DfrB DHFR is a poorly adapted catalyst and has recently been recruited to perform a novel enzymic activity in response to selective pressure.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xks1ektL0%253D&md5=0fedb66b8aabc150deee62184e4ab725
13
Strader, M. B. (2003) Identifying the Catalytic and Ligand Binding Roles of Active Site Residues in Homotetrameric R67 Dihydrofolate Reductase. Ph.D. Dissertation, University of Tennessee, Knoxville, TN.
Google Scholar
There is no corresponding record for this reference.
14
Duff, M. R., Jr., Chopra, S., Strader, M. B., Agarwal, P. K., and Howell, E. E. (2016) Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase. Biochemistry 55, 133– 145, DOI: 10.1021/acs.biochem.5b00981
Google Scholar
14
Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase
Duff, Michael R.; Chopra, Shaileja; Strader, Michael Brad; Agarwal, Pratul K.; Howell, Elizabeth E.
Biochemistry (2016), 55 (1), 133-145CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by NMR and crystallog., is disordered when bound. To explore whether smaller active site vols. (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asym. mutations that decreased the half-pore vol. by ∼35% were constructed. Only minor effects on kcat were obsd. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated crosslinking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that crosslinking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both neg. charged carboxylates in the glutamate tail were replaced with pos. charged amines. The Ki for this adduct was ∼9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFCqsbbL&md5=57afbe6aebb421eab08bf43e8b083ef5
15
Schmitzer, A. R., Lepine, F., and Pelletier, J. N. (2004) Combinatorial exploration of the catalytic site of a drug-resistant dihydrofolate reductase: creating alternative functional configurations. Protein Eng., Des. Sel. 17, 809– 819, DOI: 10.1093/protein/gzh090
Google Scholar
15
Combinatorial exploration of the catalytic site of a drug-resistant dihydrofolate reductase: creating alternative functional configurations
Schmitzer, Andreea R.; Lepine, Francois; Pelletier, Joelle N.
Protein Engineering, Design & Selection (2004), 17 (11), 809-819CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)
We have applied a global approach to enzyme active site exploration, where multiple mutations were introduced combinatorially at the active site of Type II R67 dihydrofolate reductase (R67 DHFR), creating numerous new active site environments within a const. framework. By this approach, we combinatorially modified all 16 principal amino acids that constitute the active site of this enzyme. This approach is fundamentally different from active site point mutation in that the native active site context is no longer accounted for. Among the 1536 combinatorially mutated active site variants of R67 DHFR we created, we selected and kinetically characterized three variants with highly altered active site compns. We detd. that they are of high fitness, as defined by a complex function consisting jointly of catalytic activity and resistance to trimethoprim. The kcat and KM values were similar to those for the native enzyme. The favorable Δ(ΔG) values obtained (ranging from -0.72 to -1.08 kcal/mol) suggest that, despite their complex mutational pattern, no fundamental change in the catalytic mechanism has occurred. We illustrate that combinatorial active site mutagenesis can allow for the creation of compensatory mutations that could not be predicted and thus provides a route for more extensive exploration of functional sequence space than is allowed by point mutation.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhsFSntb8%253D&md5=3563570b33610340fa17e16761e3591a
16
Bradrick, T. D., Shattuck, C., Strader, M. B., Wicker, C., Eisenstein, E., and Howell, E. E. (1996) Redesigning the quaternary structure of R67 dihydrofolate reductase. Creation of an active monomer from a tetrameric protein by quadruplication of the gene. J. Biol. Chem. 271, 28031– 28037, DOI: 10.1074/jbc.271.45.28031
Google Scholar
There is no corresponding record for this reference.
17
Dam, J. and Blondel, A. (2004) Effect of multiple symmetries on the association of R67 DHFR subunits bearing interfacial complementing mutations. Protein Sci. 13, 1– 14, DOI: 10.1110/ps.03309504
Google Scholar
17
Effect of multiple symmetries on the association of R67 DHFR subunits bearing interfacial complementing mutations
Dam, Julie; Blondel, Arnaud
Protein Science (2004), 13 (1), 1-14CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
It was shown previously that complementation could be a powerful mean to probe protein-protein interactions in the normally tetrameric R67 DHFR. Indeed, mixing complementing inactive dimeric mutants produced active heterotetramers. This approach turned a homo-oligomer into a hetero-oligomer and thus allowed the use of combinatorial assays, a subtle anal. of the assocn. forces, and a precise detn. of the equil. dissocn. consts. (KD) by titrimetry. However, for some of the complementing pairs, the exptl. data implied multiple equil. involving heterodimers, although no monomers could be detected. Thus, the reactions involved had to be identified to elaborate a suitable model to det. the KD of those pairs correctly. That model suggested that homodimers assocd. rapidly before the protomers could be redistributed in a multiple equil. system. Kinetic data confirmed that view. The assocn. data at equil. were analyzed by multiple curve fitting with all plausible combinations of parameters. This gave a confidence interval for KD that is safer than the usual 67% or 90% confidence interval. Finally, the KD of one specific reaction, the dissocn. of a heterotetramer with the relevant symmetry into two homodimers could be detd. with the relevant model for each complementing pair, although multiple equil. were present. These KD can thus be used as a set of refs. data to test and improve theor. methods such as assocn. free energy calcns.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhsFWjsg%253D%253D&md5=914326e8e1a24f83662e543b2e10df86
18
Dam, J., Rose, T., Goldberg, M. E., and Blondel, A. (2000) Complementation between dimeric mutants as a probe of dimer-dimer interactions in tetrameric dihydrofolate reductase encoded by R67 plasmid of E. coli. J. Mol. Biol. 302, 235– 250, DOI: 10.1006/jmbi.2000.4051
Google Scholar
There is no corresponding record for this reference.
19
Yachnin, B. J., Colin, D. Y., Volpato, J. P., Ebert, M., Pelletier, J. N., and Berghuis, A. M. (2011) Novel crystallization conditions for tandem variant R67 DHFR yield a wild-type crystal structure. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 67, 1316– 1322, DOI: 10.1107/S1744309111030417
Google Scholar
19
Novel crystallization conditions for tandem variant R67 DHFR yield a wild-type crystal structure
Yachnin, Brahm J.; Colin, Damien Y.; Volpato, Jordan P.; Ebert, Maximilian; Pelletier, Joelle N.; Berghuis, Albert M.
Acta Crystallographica, Section F: Structural Biology and Crystallization Communications (2011), 67 (11), 1316-1322CODEN: ACSFCL; ISSN:1744-3091. (International Union of Crystallography)
Trimethoprim is an antibiotic that targets bacterial dihydrofolate reductase (DHFR). A plasmid-encoded DHFR known as R67 DHFR provides resistance to trimethoprim in bacteria. To better understand the mechanism of this homotetrameric enzyme, a tandem dimer construct was created that linked two monomeric R67 DHFR subunits together and mutated the sequence of residues 66-69 of the first subunit from VQIY to INSF. Using a modified crystn. protocol for this enzyme that included in situ proteolysis using chymotrypsin, the tandem dimer was crystd. and the structure was solved at 1.4 Å resoln. Surprisingly, only wild-type protomers were incorporated into the crystal. Further expts. demonstrated that the variant protomer was selectively degraded by chymotrypsin, although no canonical chymotrypsin cleavage site had been introduced by these mutations.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVCnsrzK&md5=9217e44b24cc69eb9da658b3984a3fad
20
Ebert, M. C., Morley, K. L., Volpato, J. P., Schmitzer, A. R., and Pelletier, J. N. (2015) Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions. Protein Sci. 24, 495– 507, DOI: 10.1002/pro.2602
Google Scholar
20
Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions
Ebert, Maximilian C. C. J. C.; Morley, Krista L.; Volpato, Jordan P.; Schmitzer, Andreea R.; Pelletier, Joelle N.
Protein Science (2015), 24 (4), 495-507CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic, trimethoprim. R67 DHFR is genetically and structurally unrelated to Escherichia coli chromosomal DHFR and has an unusual architecture, in that 4 identical protomers form a single sym. active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active site residue has as many as 4 distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although the authors previously demonstrated that no single residue of the native active site was indispensable for function, library selection here revealed a strong bias toward maintenance of 2 native protomers per mutated tetramer. A variety of such "half-native" tetramers were shown to procure native-like catalytic activity, with similar Km values, but kcat values 5- to 33-fold lower, illustrating a high tolerance for active site substitutions. The selected variants showed a reduced thermostability (Tm ∼12° lower), which appeared to result from looser assocn. of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no active site substitutions. These results suggest that the presence of 2 native protomers in the R67 DHFR tetramer is sufficient to provide a native-like catalytic rate and thus ensure cellular proliferation.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvVOqt7w%253D&md5=7624c11254c18d3f729bf2ea719b5e6c
21
Baskakov, I. and Bolen, D. W. (1998) Forcing thermodynamically unfolded proteins to fold. J. Biol. Chem. 273, 4831– 4834, DOI: 10.1074/jbc.273.9.4831
Google Scholar
21
Forcing thermodynamically unfolded proteins to fold
Baskakov, Ilia; Bolen, D. Wayne
Journal of Biological Chemistry (1998), 273 (9), 4831-4834CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
A growing no. of biol. important proteins have been identified as fully unfolded or partially disordered. Thus, an intriguing question is whether such proteins can be forced to fold by adding solutes found in the cells of some organisms. Nature has not ignored the powerful effect that the soln. can have on protein stability and has developed the strategy of using specific solutes (called org. osmolytes) to maintain the structure and function of cellular proteins in organisms exposed to denaturing environmental stresses. Here, the authors illustrate the extraordinary capability of one such osmolyte, trimethylamine N-oxide (TMAO), to force two thermodynamically unfolded proteins to fold to native-like species having significant functional activity. In one of these examples, TMAO is shown to increase the population of the native state relative to the denatured ensemble by nearly five orders of magnitude. The ability of TMAO to force thermodynamically unstable proteins to fold presents an opportunity for structure detn. and functional studies of an important emerging class of proteins that have little or no structure without the presence of TMAO.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhs1CgtLo%253D&md5=40a68c935b335c4f86c2de8d1ffd2d88
22
Parsegian, V. A., Rand, R. P., and Rau, D. C. (2000) Osmotic stress, crowding, preferential hydration, and binding: A comparison of perspectives. Proc. Natl. Acad. Sci. U. S. A. 97, 3987– 3992, DOI: 10.1073/pnas.97.8.3987
Google Scholar
22
Osmotic stress, crowding, preferential hydration, and binding: a comparison of perspectives
Parsegian, V. A.; Rand, R. P.; Rau, D. C.
Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (8), 3987-3992CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
There has been much confusion recently about the relative merits of different approaches, osmotic stress, preferential interaction, and crowding, to describe the indirect effect of solutes on macromol. conformations and reactions. To strengthen all interpretations of measurements and to forestall further unnecessary conceptual or linguistic confusion, we show here how the different perspectives all can be reconciled. Our approach is through the Gibbs-Duhem relation, the universal constraint on the no. of ways it is possible to change the temp., pressure, and chem. potentials of the several components in any thermodynamically defined system. From this general Gibbs-Duhem equation, it is possible to see the equivalence of the different perspectives and even to show the precise identity of the more specialized equations that the different approaches use.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXislSgt70%253D&md5=158b27c5a0e496fe295b4a02385cdd34
23
Biedermannova, L. and Schneider, B. (2016) Hydration of proteins and nucleic acids: Advances in experiment and theory. A review. Biochim. Biophys. Acta, Gen. Subj. 1860, 1821– 1835, DOI: 10.1016/j.bbagen.2016.05.036
Google Scholar
23
Hydration of proteins and nucleic acids: Advances in experiment and theory. A review
Biedermannova, Lada; Schneider, Bohdan
Biochimica et Biophysica Acta, General Subjects (2016), 1860 (9), 1821-1835CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
A review. Most biol. processes involve water, and the interactions of biomols. with water affect their structure, function and dynamics. This review summarizes the current knowledge of protein and nucleic acid interactions with water, with a special focus on the biomol. hydration layer. Recent developments in both exptl. and computational methods that can be applied to the study of hydration structure and dynamics are reviewed, including software tools for the prediction and characterization of hydration layer properties. In the last decade, important advances have been made in our understanding of the factors that det. how biomols. and their aq. environment influence each other. Both exptl. and computational methods contributed to the gradually emerging consensus picture of biomol. hydration. An improved knowledge of the structural and thermodn. properties of the hydration layer will enable a detailed understanding of the various biol. processes in which it is involved, with implications for a wide range of applications, including protein-structure prediction and structure-based drug design.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XovVagurs%253D&md5=b87cb044d617b6868eec0e738f272bfc
24
Rani, P. and Biswas, P. (2015) Diffusion of Hydration Water around Intrinsically Disordered Proteins. J. Phys. Chem. B 119, 13262– 13270, DOI: 10.1021/acs.jpcb.5b07248
Google Scholar
24
Diffusion of Hydration Water around Intrinsically Disordered Proteins
Rani, Pooja; Biswas, Parbati
Journal of Physical Chemistry B (2015), 119 (42), 13262-13270CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
Hydration water dynamics around globular proteins have attracted considerable attention in the past decades. This work investigates the hydration water dynamics around partially/fully intrinsically disordered proteins (IDPs) and compares it to that of the globular proteins via mol. dynamics (MD) simulations. The translational diffusion of the hydration water is examd. by evaluating the mean-square displacement and the velocity autocorrelation function, while the rotational diffusion is probed through the dipole-dipole time correlation function. The results reveal that the translational and rotational motions of water mols. at the surface of intrinsically disordered proteins/regions are less restricted in comparison to compared to those around globular proteins/ordered regions, which is reflected in their higher diffusion coeff. and lower orientational relaxation time. The restricted mobility of hydration water in the vicinity of the protein leads to a sublinear diffusion in a heterogeneous interface. A pos. correlation between the mean no. of hydrogen bonds and the diffusion coeff. of hydration water implies higher mobility of water mols. at the surface of disordered proteins, which is due to their higher no. of hydrogen bonds. Enhanced hydration water mobility around disordered proteins/regions is also related to their higher hydration capacity, low hydrophobicity, and increased internal protein motions. Thus, we generalize that the intrinsically disordered proteins/regions are assocd. with higher hydration water mobility as compared to globular protein/ordered regions, which may help to elucidate their varied functional specificity.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFyrurnO&md5=51309a05e54693cd4ec53ee220701aca
25
Rani, P. and Biswas, P. (2015) Local Structure and Dynamics of Hydration Water in Intrinsically Disordered Proteins. J. Phys. Chem. B 119, 10858– 10867, DOI: 10.1021/jp511961c
Google Scholar
25
Local Structure and Dynamics of Hydration Water in Intrinsically Disordered Proteins
Rani, Pooja; Biswas, Parbati
Journal of Physical Chemistry B (2015), 119 (34), 10858-10867CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
Hydration water around protein surface plays a key role in structure, folding and dynamics of proteins. Intrinsically disordered proteins lack secondary and/or tertiary structure in their native state. Thus, characterizing the local structure and dynamics of hydration water around disordered proteins is challenging for both experimentalists and theoreticians. The local structure, orientation and dynamics of hydration water in the vicinity of intrinsically disordered proteins is investigated through mol. dynamics simulations. The anal. of the hydration capacity reveals that the disordered proteins have much larger binding capacity for hydration water than globular proteins. The surface and radial distribution of water mols. around the disordered proteins depict a similar trend. The local structure of the hydration water evaluated in terms of the tetrahedral order parameter, shows a higher order among the water mols. surrounding disordered proteins/regions. The residence time of water mols. clearly exhibits slow dynamics of hydration water around the surface of disordered proteins/regions as compared to globular proteins. The orientation of water mols. is found to be distinctly different for ordered and disordered proteins/regions. This anal. provides a better insight into the structure and dynamics of hydration water around disordered proteins.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmsVemtLc%253D&md5=7ebac22aa3b9ea87025dfab7fe0be5f6
26
Park, H. (1997) Creation and characterization of asymmetric mutations in R67 dihydrofolate reductases. Ph.D. Dissertation, University of Tennessee, Knoxville, TN.
Google Scholar
There is no corresponding record for this reference.
27
Stanley, C., Krueger, S., Parsegian, V. A., and Rau, D. C. (2008) Protein structure and hydration probed by SANS and osmotic stress. Biophys. J. 94, 2777– 2789, DOI: 10.1529/biophysj.107.122697
Google Scholar
27
Protein structure and hydration probed by SANS and osmotic stress
Stanley, Christopher; Krueger, Susan; Parsegian, V. Adrian; Rau, Donald C.
Biophysical Journal (2008), 94 (7), 2777-2789CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
Interactions governing protein folding, stability, recognition, and activity are mediated by hydration. Here, we use small-angle neutron scattering (SANS) coupled with osmotic stress to investigate the hydration of two proteins, lysozyme and guanylate kinase (GK), in the presence of solutes. By taking advantage of the neutron contrast variation that occurs upon addn. of these solutes, the no. of protein-assocd. (solute-excluded) water mols. can be estd. from changes in both the zero-angle scattering intensity and the radius of gyration. Poly(ethylene glycol) exclusion varies with mol. wt. This sensitivity can be exploited to probe structural features such as the large internal GK cavity. For GK, small-angle neutron scattering is complemented by isothermal titrn. calorimetry with osmotic stress to also measure hydration changes accompanying ligand binding. These results provide a framework for studying other biomol. systems and assemblies using neutron scattering together with osmotic stress.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjslCjsrk%253D&md5=b3ec7dd2048fd5f6e8e58a3820b81be1
28
Arnold, O., Bilheux, J. C., Borreguero, J. M., Buts, A., Campbell, S. I., Chapon, L., Doucet, M., Draper, N., Ferraz Leal, R., Gigg, M. A., Lynch, V. E., Markvardsen, A., Mikkelson, D. J., Mikkelson, R. L., Miller, R., Palmen, K., Parker, P., Passos, G., Perring, T. G., Peterson, P. F., Ren, S., Reuter, M. A., Savici, A. T., Taylor, J. W., Taylor, R. J., Tolchenov, R., Zhou, W., and Zikovsky, J. (2014) Mantid-Data analysis and visualization package for neutron scattering and mu SR experiments. Nucl. Instrum. Methods Phys. Res., Sect. A 764, 156– 166, DOI: 10.1016/j.nima.2014.07.029
Google Scholar
28
Mantid-Data analysis and visualization package for neutron scattering and μ SR experiments
Arnold, O.; Bilheux, J. C.; Borreguero, J. M.; Buts, A.; Campbell, S. I.; Chapon, L.; Doucet, M.; Draper, N.; Ferraz Leal, R.; Gigg, M. A.; Lynch, V. E.; Markvardsen, A.; Mikkelson, D. J.; Mikkelson, R. L.; Miller, R.; Palmen, K.; Parker, P.; Passos, G.; Perring, T. G.; Peterson, P. F.; Ren, S.; Reuter, M. A.; Savici, A. T.; Taylor, J. W.; Taylor, R. J.; Tolchenov, R.; Zhou, W.; Zikovsky, J.
Nuclear Instruments & Methods in Physics Research, Section A: Accelerators, Spectrometers, Detectors, and Associated Equipment (2014), 764 (), 156-166CODEN: NIMAER; ISSN:0168-9002. (Elsevier B.V.)
The Mantid framework is a software soln. developed for the anal. and visualization of neutron scattering and muon spin measurements. The framework is jointly developed by software engineers and scientists at the ISIS Neutron and Muon Facility and the Oak Ridge National Lab. The objectives, functionality and novel design aspects of Mantid are described.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlGntrfL&md5=9a34bb9e366fca0e39091a5c4016ccd7
29
Svergun, D. I. (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria.. J. Appl. Crystallogr. 25, 495– 503, DOI: 10.1107/S0021889892001663
Google Scholar
29
Determination of the regularization parameter in indirect-transform methods using perceptual criteria
Svergun, D. I.
Journal of Applied Crystallography (1992), 25 (4), 495-503CODEN: JACGAR; ISSN:1600-5767. (International Union of Crystallography)
A method is proposed for the detn. of the optimum value of the regularization parameter (Lagrange multiplier) when applying indirect transform techniques in small-angle scattering data anal. The method is based on perceptual criteria of what is the best soln. A set of simple criteria is used to construct a total est. describing the quality of the soln. Maximization of the total est. is straightforward. Model computations show the effectiveness of the technique. The method is implemented in the program GNOM.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslaks7vF&md5=98cf53dc2febca54bbbd505f698e1269
30
Whitten, A. E., Cai, S., and Trewhella, J. (2008) MULCh: modules for the analysis of small-angle neutron contrast variation data from biomolecular assemblies. J. Appl. Crystallogr. 41, 222– 226, DOI: 10.1107/S0021889807055136
Google Scholar
30
MULCh: modules for the analysis of small-angle neutron contrast variation data from biomolecular assemblies
Whitten, Andrew E.; Cai, Shuzhi; Trewhella, Jill
Journal of Applied Crystallography (2008), 41 (1), 222-226CODEN: JACGAR; ISSN:0021-8898. (International Union of Crystallography)
Small-angle neutron scattering with contrast variation can fill important gaps in our understanding of biomol. assemblies, providing constraints that can aid in the construction of mol. models and in subsequent model refinements. This paper describes the implementation of simple tools for analyzing neutron contrast variation data, accessible via a user-friendly web-based interface (http://www.mmb.usyd.edu.au/NCVWeb/). There are three modules accessible from the website to analyze neutron contrast variation data from bimol. complexes. The first module, Contrast, computes neutron contrasts of each component of the complex required by the other two modules; the second module, Rg, analyses the contrast dependence of the radii of gyration to yield information relating to the size and disposition of each component in the complex; and the third, Compost, decomps. the contrast variation series into composite scattering functions, which contain information regarding the shape of each component of the complex, and their orientation with respect to each other.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmvFymtw%253D%253D&md5=362603873968ba4922479a40906d85c7
31
Curtis, J. E., Raghunandan, S., Nanda, H., and Krueger, S. (2012) SASSIE: A program to study intrinsically disordered biological molecules and macromolecular ensembles using experimental scattering restraints,. Comput. Phys. Commun. 183, 382– 389, DOI: 10.1016/j.cpc.2011.09.010
Google Scholar
31
SASSIE: A program to study intrinsically disordered biological molecules and macromolecular ensembles using experimental scattering restraints
Curtis, Joseph E.; Raghunandan, Sindhu; Nanda, Hirsh; Krueger, Susan
Computer Physics Communications (2012), 183 (2), 382-389CODEN: CPHCBZ; ISSN:0010-4655. (Elsevier B.V.)
A program to construct ensembles of biomol. structures that are consistent with exptl. scattering data are described. Specifically, we generate an ensemble of biomol. structures by varying sets of backbone dihedral angles that are then filtered using exptl. detd. restraints to rapidly det. structures that have scattering profiles that are consistent with scattering data. We discuss an application of these tools to predict a set of structures for the HIV-1 Gag protein, an intrinsically disordered protein, that are consistent with small-angle neutron scattering exptl. data. We have assembled these algorithms into a program called SASSIE for structure generation, visualization, and anal. of intrinsically disordered proteins and other macromol. ensembles using neutron and X-ray scattering restraints. Open source software to generate structures of disordered biol. mols. that subsequently allow for the comparison of computational and exptl. results is limiting the use of scattering resources. Starting with an all atom model of a protein, for example, users can input regions to vary dihedral angles, ensembles of structures can be generated. Addnl., simple two-body rigid-body rotations are supported with and without disordered regions. Generated structures can then be used to calc. small-angle scattering profiles which can then be filtered against exptl. detd. data. Filtered structures can be visualized individually or as an ensemble using d. plots. In the modular and expandable program framework the user can easily access our subroutines and structural coordinates can be easily obtained for study using other computational physics methods. The distribution file for this program is over 159 Mbytes and therefore is not delivered directly when download or Email is requested. Instead an html file giving details of how the program can be obtained is sent. Running time varies depending on application. Typically 10 min to 24 h depending on the no. of generated structures.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsV2is7vJ&md5=ad02c0b0220a6da5de20deaab3b1bee7
32
Webb, B. and Sali, A. (2014) Comparative Protein Structure Modeling Using MODELLER. Current Protocols in Bioinformatics 47, 5.6.1– 5.6.32, DOI: 10.1002/0471250953.bi0506s47
Google Scholar
There is no corresponding record for this reference.
33
Berendsen, H. J. C., Grigera, J. R., and Straatsma, T. P. (1987) The missing term in effective pair potentials. J. Phys. Chem. 91, 6269– 6271, DOI: 10.1021/j100308a038
Google Scholar
33
The missing term in effective pair potentials
Berendsen, H. J. C.; Grigera, J. R.; Straatsma, T. P.
Journal of Physical Chemistry (1987), 91 (24), 6269-71CODEN: JPCHAX; ISSN:0022-3654.
Effective pair potentials used for simulations of polar liqs. include the av. effects of polarization. Such potentials are generally adjusted to produce the exptl. heat of vaporization. It has not been recognized before that the self-energy term inherent in any polarizable model should be included in effective pair potentials as well. Inclusion of the self-energy correction with a consequent reparametrization of the simple point charge model of water yields an improvement of the effective pair potential for water, as exemplified by d., radial distribution functions, and diffusion const.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXmt1els7w%253D&md5=6668667f6252092fc001ae8d422ebb94
34
Ramanathan, A., Savol, A. J., Langmead, C. J., Agarwal, P. K., and Chennubhotla, C. S. (2011) Discovering conformational sub-states relevant to protein function. PLoS One 6, e15827 DOI: 10.1371/journal.pone.0015827
Google Scholar
34
Discovering conformational sub-states relevant to protein function
Ramanathan, Arvind; Savol, Andrej J.; Langmead, Christopher J.; Agarwal, Pratul K.; Chennubhotla, Chakra S.
PLoS One (2011), 6 (1), e15827CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
Background: Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant. However, low populations in these sub-states and the transient nature of conformational transitions between these sub-states present significant challenges for their identification and characterization. Methods and Findings: To overcome these challenges we have developed a new computational technique, quasi-anharmonic anal. (QAA). QAA utilizes higher-order statistics of protein motions to identify sub-states in the conformational landscape. Further, the focus on anharmonicity allows identification of conformational fluctuations that enable transitions between sub-states. QAA applied to equil. simulations of human ubiquitin and T4 lysozyme reveals functionally relevant sub-states and protein motions involved in mol. recognition. In combination with a reaction pathway sampling method, QAA characterizes conformational sub-states assocd. with cis/trans peptidyl-prolyl isomerization catalyzed by the enzyme cyclophilin A. In these 3 proteins, QAA allows identification of conformational sub-states, with crit. structural and dynamical features relevant to protein function. Conclusions: Overall, QAA provides a novel framework to intuitively understand the biophys. basis of conformational diversity and its relevance to protein function.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhvVKksro%253D&md5=7ecb8360e065745b1cdae3f4b23968d8
35
Case, D. A., Babin, V., Berryman, J. T., Betz, R. M., Cai, Q., Cerutti, D. S., Cheatham, T. E., III, Darden, T. A., Duke, R. E., Gohlke, H., Goetz, A. W., Gusarov, S., Homeyer, N., Janowski, P., Kaus, J., Kolossváry, I., Kovalenko, A., Lee, T. S., LeGrand, S., Luchko, T., Luo, R., Madej, B., Merz, K. M., Paesani, F., Roe, D. R., Roitberg, A., Sagui, C., Salomon-Ferrer, R., Seabra, G., Simmerling, C. L., Smith, W., Walker, R. C., Wang, J., Wolf, R. M., Wu, X., and Kollman, P. A. (2014) AMBER14, University of California, San Francisco.
Google Scholar
There is no corresponding record for this reference.
36
Bradrick, T. D., Beechem, J. M., and Howell, E. E. (1996) Unusual binding stoichiometries and cooperativity are observed during binary and ternary complex formation in the single active pore of R67 dihydrofolate reductase, a D₂ symmetric protein. Biochemistry 35, 11414– 11424, DOI: 10.1021/bi960205d
Google Scholar
36
Unusual Binding Stoichiometries and Cooperativity Are Observed during Binary and Ternary Complex Formation in the Single Active Pore of R67 Dihydrofolate Reductase, a D2 Symmetric Protein
Bradrick, Thomas D.; Beechem, Joseph M.; Howell, Elizabeth E.
Biochemistry (1996), 35 (35), 11414-11424CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
R67 dihydrofolate reductase (DHFR) is an R-plasmid-encoded enzyme that confers resistance to the antibacterial drug, trimethoprim. This DHFR variant is not homologous in either sequence or structure to chromosomal DHFRs. A recent crystal structure of the active tetrameric species describes a single active site pore that traverses the length of the protein (Narayana et al., 1995). Related sites (due to a 222 symmetry element at the center of the active site pore) are used for binding of ligands, i.e., each half-pore can accommodate either the substrate, dihydrofolate, or the cofactor, NADPH, although dihydrofolate and NADPH are bound differently. Ligand binding in R67 DHFR was evaluated using time-resolved fluorescence anisotropy and isothermal titrn. calorimetry techniques. Under binary complex conditions, two mols. of either NADPH, folate, dihydrofolate, or N10 propargyl-5,8-dideazafolate (CB3717) can be bound. Binding of NADPH displays neg. cooperativity, binding of either folate or dihydrofolate shows pos. cooperativity, and binding of CB3717 shows two identical sites. Any asymmetry introduced by binding of one ligand is proposed to induce the cooperativity assocd. with binding of the second ligand. Evaluation of ternary complex formation demonstrates that one mol. of folate binds to a 1:1 mixt. of R67 DHFR + NADPH. These binding results indicate a max. of two ligands bind in the pore. A mechanism describing catalysis is proposed that is consistent with the binding results.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XkvVGqsbc%253D&md5=f85af8ec7de91eb41c60c55aae8604a9
37
Lin, L. N., Brandts, J. F., Brandts, J. M., and Plotnikov, V. (2002) Determination of the volumetric properties of proteins and other solutes using pressure perturbation calorimetry. Anal. Biochem. 302, 144– 160, DOI: 10.1006/abio.2001.5524
Google Scholar
37
Determination of the volumetric properties of proteins and other solutes using pressure perturbation calorimetry
Lin, Lung-Nan; Brandts, John F.; Brandts, J. Michael; Plotnikov, Valerian
Analytical Biochemistry (2002), 302 (1), 144-160CODEN: ANBCA2; ISSN:0003-2697. (Academic Press)
Pressure perturbation calorimetry is a new technique that measures the heat change in a soln. that results when the pressure above the soln. is changed. When used in a differential calorimeter contg. a dil. soln. of solute in the sample cell and the corresponding buffer in the ref. cell, the measured differential heat can be used to calc. the thermal coeff. of expansion of the partial vol. of the solute,. For proteins in dil. aq. soln., is dominated by a temp.-dependent contribution arising from the interaction of protein groups with water at the protein-solvent interface. This arises due to the effect of the protein groups on the hydrogen-bonded structure of water, and thereby clearly differentiates between structure-making hydrophobic groups and structure-breaking hydrophilic groups. This solvation contribution to can be accentuated in solvents having more structure (deuterium oxide) than water and attenuated in solvents having less structure (2.8 M guanidinium sulfate). Six different proteins (chymotrypsinogen, pepsinogen, lysozyme, bovine pancreatic trypsin inhibitor, RNase A, and T4 lysozyme) were examd. carefully by this technique, allowing ests. of various volumetric parameters including the vol. change resulting from thermal unfolding of each protein. For RNase A, results obtained in both water and deuterium oxide led to an est. of the accessible surface area of the native protein of ∼45% relative to the fully reduced unfolded protein. Also, it was also found that ligand binding to RNase A led to changes in, suggesting a burial of some surface area in the ligand-protein complex. (c) 2002 Academic Press.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhtFels78%253D&md5=15248e92350b9659d4c7934605921e2f
38
Mitra, L., Smolin, N., Ravindra, R., Royer, C., and Winter, R. (2006) Pressure perturbation calorimetric studies of the solvation properties and the thermal unfolding of proteins in solution–experiments and theoretical interpretation. Phys. Chem. Chem. Phys. 8, 1249– 1265, DOI: 10.1039/b516608j
Google Scholar
There is no corresponding record for this reference.
39
Keller, S., Vargas, C., Zhao, H., Piszczek, G., Brautigam, C. A., and Schuck, P. (2012) High-precision isothermal titration calorimetry with automated peak-shape analysis. Anal. Chem. 84, 5066– 5073, DOI: 10.1021/ac3007522
Google Scholar
39
High-Precision Isothermal Titration Calorimetry with Automated Peak-Shape Analysis
Keller, Sandro; Vargas, Carolyn; Zhao, Huaying; Piszczek, Grzegorz; Brautigam, Chad A.; Schuck, Peter
Analytical Chemistry (Washington, DC, United States) (2012), 84 (11), 5066-5073CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
Isothermal titrn. calorimetry (ITC) is a powerful classical method that enables researchers in many fields to study the thermodn. of mol. interactions. Primary ITC data comprise the temporal evolution of differential power reporting the heat of reaction during a series of injections of aliquots of a reactant into a sample cell. By integration of each injection peak, an isotherm can be constructed of total changes in enthalpy as a function of changes in soln. compn., which is rich in thermodn. information on the reaction. However, the signals from the injection peaks are superimposed by the stochastically varying time-course of the instrumental baseline power, limiting the precision of ITC isotherms. The authors describe a method for automated peak assignment based on peak-shape anal. via singular value decompn. in combination with detailed least-squares modeling of local pre- and post- injection baselines. This approach can effectively filter out contributions of short-term noise and adventitious events in the power trace. This method also provides, for the first time, statistical error ests. for the individual isotherm data points. In turn, this results in improved detection limits for high-affinity or low-enthalpy binding reactions and significantly higher precision of the derived thermodn. parameters.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmtVGlsbs%253D&md5=bb157e956fa9cef94a88974d733cad1b
40
Timson, M. J., Duff, M. R., Jr., Dickey, G., Saxton, A. M., Reyes-De-Corcuera, J. I., and Howell, E. E. (2013) Further studies on the role of water in R67 dihydrofolate reductase. Biochemistry 52, 2118– 2127, DOI: 10.1021/bi301544k
Google Scholar
40
Further Studies on the Role of Water in R67 Dihydrofolate Reductase
Timson, Mary Jane; Duff, Michael R.; Dickey, Greyson; Saxton, Arnold M.; Reyes-De-Corcuera, Jose I.; Howell, Elizabeth E.
Biochemistry (2013), 52 (12), 2118-2127CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
Previous osmotic pressure studies of two nonhomologous dihydrofolate reductase (DHFR) enzymes found tighter binding of the NADP cofactor upon addn. of neutral osmolytes. This result is consistent with water release accompanying binding. In contrast, osmotic stress studies found weaker binding of the dihydrofolate (DHF) substrate for both type I and type II DHFRs in the presence of osmolytes; this observation can be explained if dihydrofolate interacts with osmolytes and shifts the equil. from the enzyme-bound state toward the unbound substrate. NMR expts. support this hypothesis, finding that osmolytes interact with dihydrofolate. To consider binding without added osmolytes, a high-pressure approach was used. In this study, the type II enzyme, R67 DHFR, was subjected to high hydrostatic pressure (HHP). Both enzyme activity and fluorescence measurements find the protein tolerates pressures up to 200 MPa. Binding of the cofactor to R67 DHFR weakens with increasing pressure, and a pos. assocn. vol. of 11.4 ± 0.5 cm3/mol was measured. Addnl., an activation vol. of 3.3 ± 0.5 cm3/mol describing kcat/Km(DHF) was detd. from progress curve anal. Results from these HHP expts. suggest water release accompanies binding of both the cofactor and DHF to R67 DHFR. In an addnl. set of expts., isothermal titrn. calorimetry studies in H2O and D2O find that water reorganization dominates the enthalpy assocd. with binding of DHF to R67 DHFR·NADP+, while no obvious effects occur for cofactor binding. The combined results indicate that water plays an active role in ligand binding to R67 DHFR.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjtlGht7c%253D&md5=222ea1b1b4967fa95385d5cf63fa4ade
41
Receveur-Brechot, V. and Durand, D. (2012) How random are intrinsically disordered proteins? A small angle scattering perspective,. Curr. Protein Pept. Sci. 13, 55– 75, DOI: 10.2174/138920312799277901
Google Scholar
41
How random are intrinsically disordered proteins? A small angle scattering perspective
Receveur-Brechot, Veronique; Durand, Dominique
Current Protein and Peptide Science (2012), 13 (1), 55-75CODEN: CPPSCM; ISSN:1389-2037. (Bentham Science Publishers Ltd.)
A review. While the crucial role of intrinsically disordered proteins (IDPs) in the cell cycle is now recognized, deciphering their mol. mode of action at the structural level still remains highly challenging and requires a combination of many biophys. approaches. Among them, small angle X-ray scattering (SAXS) has been extremely successful in the last decade and has become an indispensable technique for addressing many of the fundamental questions regarding the activities of IDPs. After introducing some exptl. issues specific to IDPs and in relation to the latest tech. developments, this article presents the interest of the theory of polymer physics to evaluate the flexibility of fully disordered proteins. The different strategies to obtain 3-dimensional models of IDPs, free in soln. and assocd. in a complex, are then reviewed. Indeed, recent computational advances have made it possible to readily ext. max. information from the scattering curve with a special emphasis on highly flexible systems, such as multidomain proteins and IDPs. Furthermore, integrated computational approaches now enable the generation of ensembles of conformers to translate the unique flexible characteristics of IDPs by taking into consideration the constraints of more and more various complementary expt. In particular, a combination of SAXS with high-resoln. techniques, such as x-ray crystallog. and NMR, allows us to provide reliable models and to gain unique structural insights about the protein over multiple structural scales. The latest neutron scattering expts. also promise new advances in the study of the conformational changes of macromols. involving more complex systems.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkvFamtLw%253D&md5=9bb9c34254af874c361317049c577d3c
42
Svergun, D. I., Richard, S., Koch, M. H., Sayers, Z., Kuprin, S., and Zaccai, G. (1998) Protein hydration in solution: experimental observation by x-ray and neutron scattering. Proc. Natl. Acad. Sci. U. S. A. 95, 2267– 2272, DOI: 10.1073/pnas.95.5.2267
Google Scholar
42
Protein hydration in solution: experimental observation by x-ray and neutron scattering
Svergun, D. I.; Richard, S.; Koch, M. H. J.; Sayers, Z.; Kuprin, S.; Zaccai, G.
Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (5), 2267-2272CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
The structure of the protein-solvent interface is the subject of controversy in theor. studies and requires direct exptl. characterization. Three proteins with known at. resoln. crystal structure (lysozyme, Escherichia coli thioredoxin reductase, and protein R1 of E. coli ribonucleotide reductase) were investigated in parallel by x-ray and neutron scattering in H2O and D2O solns. The anal. of the protein-solvent interface is based on the significantly different contrasts for the protein and for the hydration shell. The results point to the existence of a first hydration shell with an av. d. ≈10% larger than that of the bulk solvent in the conditions studied. Comparisons with the results of other studies suggest that this may be a general property of aq. interfaces.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhsleitrw%253D&md5=415ed528ad555452e1b8fb119353c1af
43
Rambo, R. P. and Tainer, J. A. (2013) Accurate assessment of mass, models and resolution by small-angle scattering. Nature 496, 477– 481, DOI: 10.1038/nature12070
Google Scholar
43
Accurate assessment of mass, models and resolution by small-angle scattering
Rambo, Robert P.; Tainer, John A.
Nature (London, United Kingdom) (2013), 496 (7446), 477-481CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
Modern small-angle scattering (SAS) expts. with X-rays or neutrons provide a comprehensive, resoln.-limited observation of the thermodn. state. However, methods for evaluating mass and validating SAS-based models and resoln. have been inadequate. Here we define the vol. of correlation, Vc, a SAS invariant derived from the scattered intensities that is specific to the structural state of the particle, but independent of concn. and the requirements of a compact, folded particle. We show that Vc defines a ratio, QR, that dets. the mol. mass of proteins or RNA ranging from 10 to 1,000 kilodaltons. Furthermore, we propose a statistically robust method for assessing model-data agreements (χ2free) akin to cross-validation. Our approach prevents over-fitting of the SAS data and can be used with a newly defined metric, RSAS, for quant. evaluation of resoln. Together, these metrics (Vc, QR, χ2free and RSAS) provide anal. tools for unbiased and accurate macromol. structural characterizations in soln.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXms1Wgu7o%253D&md5=ca998ec661eaa9015e93c4f52c083bce
44
Santner, A. A., Croy, C. H., Vasanwala, F. H., Uversky, V. N., Van, Y. Y., and Dunker, A. K. (2012) Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression. Biochemistry 51, 7250– 7262, DOI: 10.1021/bi300653m
Google Scholar
44
Sweeping Away Protein Aggregation with Entropic Bristles: Intrinsically Disordered Protein Fusions Enhance Soluble Expression
Santner, Aaron A.; Croy, Carrie H.; Vasanwala, Farha H.; Uversky, Vladimir N.; Van, Ya-Yue J.; Dunker, A. Keith
Biochemistry (2012), 51 (37), 7250-7262CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both a large favorable surface area for water interactions and large excluded vols. around the partner. By extending away from the partner and sweeping out large mols., EBs can allow the target protein to fold free from interference. Using both naturally occurring and artificial polypeptides, the authors demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have a low level of sequence complexity and a high net charge but are diversified by distinctive amino acid compns. and lengths. Using 6xHis fusions as controls, sol. protein expression enhancements from 65% (EB60A) to 100% (EB250) were obsd. for a 20-protein portfolio. Addnl., these EBs were able to more effectively solubilize targets compared to frequently used fusions such as maltose-binding protein, glutathione S-transferase, thioredoxin, and N use substance A. Finally, although these EBs possess very distinct physiochem. properties, they did not perturb the structure, conformational stability, or function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically disordered fusions and presents a promising technol. and complementary resource for researchers attempting to solubilize recalcitrant proteins.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1Kitr3F&md5=abf5e6e0125744b920386c7922a0588b
45
Fried, M. G., Stickle, D. F., Smirnakis, K. V., Adams, C., MacDonald, D., and Lu, P. (2002) Role of hydration in the binding of lac repressor to DNA. J. Biol. Chem. 277, 50676– 50682, DOI: 10.1074/jbc.M208540200
Google Scholar
45
Role of Hydration in the Binding of lac Repressor to DNA
Fried, Michael G.; Stickle, Douglas F.; Smirnakis, Karen Vossen; Adams, Claire; MacDonald, Douglas; Lu, Ponzy
Journal of Biological Chemistry (2002), 277 (52), 50676-50682CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
The osmotic stress technique was used to measure changes in macromol. hydration that accompany binding of wild-type Escherichia coli lactose (lac) repressor to its regulatory site (operator O1) in the lac promoter and its transfer from site O1 to nonspecific DNA. Binding at O1 is accompanied by the net release of 260±32 water mols. If all are released from macromol. surfaces, this result is consistent with a net redn. of solvent-accessible surface area of 2370±550 Å. This area is only slightly smaller than the macromol. interface calcd. for a cryst. repressor dimer-O1 complex but is significantly smaller than that for the corresponding complex with the sym. optimized Osym operator. The transfer of repressor from site O1 to nonspecific DNA is accompanied by the net uptake of 93±10 water mols. Together these results imply that formation of a nonspecific complex is accompanied by the net release of 165±43 water mols. The enhanced stabilities of repressor-DNA complexes with increasing osmolality may contribute to the ability of Escherichia coli cells to tolerate dehydration and/or high external salt concns.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xps12nt7o%253D&md5=a3f2035c2b60dd88e07c86ae3a440cb7
46
Tsodikov, O. V., Record, M. T., Jr., and Sergeev, Y. V. (2002) Novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature. J. Comput. Chem. 23, 600– 609, DOI: 10.1002/jcc.10061
Google Scholar
46
Novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature
Tsodikov, Oleg V.; Record, M. Thomas, Jr.; Sergeev, Yuri V.
Journal of Computational Chemistry (2002), 23 (6), 600-609CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
New computer programs, SurfRace and FastSurf, perform fast calcns. of the solvent accessible and mol. (solvent excluded) surface areas of macromols. Program SurfRace also calcs. the areas of cavities inaccessible from the outside. We introduce the definition of av. curvature of mol. surface and calc. av. mol. surface curvatures for each atom in a structure. All surface area and curvature calcns. are analytic and therefore yield exact values of these quantities. High calcn. speed of this software is achieved primarily by avoiding computationally expensive math. procedures wherever possible and by efficient handling of surface data structures. The programs are written initially in the language C for PCs running Windows 2000/98/NT, but their code is portable to other platforms with only minor changes in input-output procedures. The algorithm is robust and does not ignore either multiplicity or degeneracy of at. overlaps. Fast, memory-efficient and robust execution make this software attractive for applications both in computationally expensive energy minimization algorithms, such as docking or mol. dynamics simulations, and in stand-alone surface area and curvature calcns.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjtFKrtLY%253D&md5=52b6b7f3c22ecb6d541167ab361501b1
47
Zhuang, P., Eisenstein, E., and Howell, E. E. (1994) Equilibrium folding studies of tetrameric R67 dihydrofolate reductase. Biochemistry 33, 4237– 4244, DOI: 10.1021/bi00180a018
Google Scholar
47
Equilibrium folding studies of tetrameric R67 dihydrofolate reductase
Zhuang, Ping; Eisenstein, Edward; Howell, Elizabeth E.
Biochemistry (1994), 33 (14), 4237-44CODEN: BICHAW; ISSN:0006-2960.
R67 dihydrofolate reductase (DHFR) is an R-plasmid encoded enzyme that confers resistance to the antibacterial drug trimethoprim. This enzyme is not homologous in sequence or structure to chromosomal DHFRs. Equil. folding of tetrameric R67 DHFR was studied and found to be fully reversible. Formation of an inactive intermediate was assayed by loss of enzyme activity. Denaturation of the intermediate was monitored by concurrent changes in fluorescence and CD signals. Both transitions are protein concn. dependent. A simple model fitting these data is tetramer ↹ 2 dimers ↹ 4 unfolded monomers. No evidence for folded monomer was found. Global fitting of all the folding data yielded a ΔGH2O of -9.63 kcal/mol for the initial transition and a ΔGH2O of -12.35 kcal/mol for the second transition. In addn., thermal unfolding of tetrameric R67 DHFR was found to be reversible. A folding intermediate also occurred during thermal unfolding as evidenced by the asym. endotherms and a ΔHcalorimetric/ΔHvan't Hoff ratio of 2.1.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXivFarsLw%253D&md5=e3972a1fba7dbd915381fd226449d670
48
Permyakov, S. (2012) Differential Scanning Microcalorimetry of Intrinsically Disordered Proteins. In Intrinsically Disordered Protein Analysis, Vol. 2, Methods and Experimental Tools (Uversky, V. L., and Dunker, A. K., Eds.) pp 283– 296, Springer, Dordrecht, The Netherlands.
Google Scholar
There is no corresponding record for this reference.
49
Permyakov, S. E., Bakunts, A. G., Denesyuk, A. I., Knyazeva, E. L., Uversky, V. N., and Permyakov, E. A. (2008) Apo-parvalbumin as an intrinsically disordered protein. Proteins: Struct., Funct., Genet. 72, 822– 836, DOI: 10.1002/prot.21974
Google Scholar
There is no corresponding record for this reference.
50
Parsegian, V. A., Rand, R. P., and Rau, D. C. (1995) Macromolecules and water: probing with osmotic stress. Methods Enzymol. 259, 43– 94, DOI: 10.1016/0076-6879(95)59039-0
Google Scholar
50
Macromolecules and water: probing with osmotic stress
Parsegian, V. Adrian; Rand, R. Peter; Rau, Donald C.
Methods in Enzymology (1995), 259 (Energetics of Biological Macromolecules), 43-94CODEN: MENZAU; ISSN:0076-6879. (Academic)
An intuitive picture of osmotic stress on macromols. is given. The authors also illustrate the method as it is applied to 4 kinds of processes: (1)ionic channel opening/closing, (2)enzyme/substrate assocn. and turnover, (3)mol. binding, and (4)long-range interaction.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xht1entbw%253D&md5=1c37a31f8ecfbc0cb05cf27e508d131d
51
Royer, C. A. (2002) Revisiting volume changes in pressure-induced protein unfolding. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1595, 201– 209, DOI: 10.1016/S0167-4838(01)00344-2
Google Scholar
51
Revisiting volume changes in pressure-induced protein unfolding
Royer, Catherine A.
Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (2002), 1595 (1-2), 201-209CODEN: BBAEDZ; ISSN:0167-4838. (Elsevier B.V.)
A review. It has long been known that the application of hydrostatic pressure generally leads to the unfolding of proteins. Despite a relatively large no. of reports in the literature over the past few decades, there has been great confusion over the sign and magnitude as well as the fundamental factors contributing to vol. effects in protein conformational transitions. It is the goal of this review to present and discuss the results obtained concerning the sign and magnitude of the vol. changes accompanying the unfolding of proteins. The vast majority of cases point to a significant decrease in vol. upon unfolding. Nonetheless, there is evidence that, due to differences in the thermal expansivity of the folded and unfolded states of proteins reported in a half dozen manuscripts, that the sign of the vol. change may become pos. at higher temps.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xjt1Sku7g%253D&md5=0a0bd5797b5bc69072f5cdb57b0a931a
52
Zhai, Y., Okoro, L., Cooper, A., and Winter, R. (2011) Applications of pressure perturbation calorimetry in biophysical studies. Biophys. Chem. 156, 13– 23, DOI: 10.1016/j.bpc.2010.12.010
Google Scholar
There is no corresponding record for this reference.
53
Ravindra, R., Royer, C., and Winter, R. (2004) Pressure perturbation calorimetic studies of the solvation properties and the thermal unfolding of Staphylococcal nuclease. Phys. Chem. Chem. Phys. 6, 1952– 1961, DOI: 10.1039/b314172a
Google Scholar
There is no corresponding record for this reference.
54
Eisenberg, D., Weiss, R. M., Terwilliger, T. C., and Wilcox, W. (1982) Hydrophobic moments and protein structure. Faraday Symp. Chem. Soc. 17, 109– 120, DOI: 10.1039/fs9821700109
Google Scholar
There is no corresponding record for this reference.
55
Wang, A. and Bolen, D. W. (1997) A naturally occurring protective system in urea-rich cells: mechanism of osmolyte protection of proteins against urea denaturation,. Biochemistry 36, 9101– 9108, DOI: 10.1021/bi970247h
Google Scholar
There is no corresponding record for this reference.
56
Bhojane, P. P., Duff, M. R., Jr., Bafna, K., Rimmer, G. P., Agarwal, P. K., and Howell, E. E. (2016) Aspects of Weak Interactions between Folate and Glycine Betaine. Biochemistry 55, 6282– 6294, DOI: 10.1021/acs.biochem.6b00873
Google Scholar
56
Aspects of Weak Interactions between Folate and Glycine Betaine
Bhojane, Purva P.; Duff, Michael R.; Bafna, Khushboo; Rimmer, Gabriella P.; Agarwal, Pratul K.; Howell, Elizabeth E.
Biochemistry (2016), 55 (45), 6282-6294CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
Folic acid, or vitamin B9, is an important compd. in 1C metab. Previous studies have found weaker binding of dihydrofolate to dihydrofolate reductase in the presence of osmolytes. In other words, osmolytes are more difficult to remove from the dihydrofolate solvation shell than water; this shifts the equil. toward the free ligand and protein species. Here, vapor pressure osmometry was used to explore the interaction of folate with the model osmolyte, glycine betaine. This method yielded a preferential interaction potential (μ23/RT value). This value was concn.-dependent as folate dimerizes. The μ23/RT value also tracked the deprotonation of folate's N3-O4 keto-enol group, yielding a pKa of 8.1. To det. which folate atoms interact most strongly with betaine, the interaction of heterocyclic arom. compds. (as well as other small mols.) with betaine was monitored. Using an accessible surface area approach coupled with osmometry measurements, deconvolution of the μ23/RT value into α values for atom types was achieved. This allowed prediction of μ23/RT values for larger mols. such as folate. Mol. dynamics simulations of folate showed a variety of structures from extended to L-shaped. These conformers possessed μ23/RT values from -0.18 to 0.09 m-1, where a neg. value indicates a preference for solvation by betaine and a pos. value indicates a preference for water. This range of values was consistent with values obsd. in osmometry and soly. expts. As the av. predicted folate μ23/RT value was near zero, this indicated that folate interacts almost equally well with betaine and water. Specifically, the glutamate tail prefers to interact with water while the arom. rings prefer betaine. In general, the more protonated species in this small mol. survey interacted better with betaine as they provided a source of H atoms (betaine is not a H-bond donor). Upon deprotonation of the small mol., the preference swings toward water interaction due to its H-bond donating capacities.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslagsbjP&md5=88ddfed96ee7bf5fa074f4309684df52
57
Capp, M. W., Pegram, L. M., Saecker, R. M., Kratz, M., Riccardi, D., Wendorff, T., Cannon, J. G., and Record, M. T., Jr (2009) Interactions of the osmolyte glycine betaine with molecular surfaces in water: thermodynamics, structural interpretation, and prediction of m-values. Biochemistry 48, 10372– 10379, DOI: 10.1021/bi901273r
Google Scholar
57
Interactions of the Osmolyte Glycine Betaine with Molecular Surfaces in Water: Thermodynamics, Structural Interpretation, and Prediction of m-Values
Capp, Michael W.; Pegram, Laurel M.; Saecker, Ruth M.; Kratz, Megan; Riccardi, Demian; Wendorff, Timothy; Cannon, Jonathan G.; Record, M. Thomas
Biochemistry (2009), 48 (43), 10372-10379CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
Noncovalent self-assembly of biopolymers is driven by mol. interactions between functional groups on complementary biopolymer surfaces, replacing interactions with water. Since individually these interactions are comparable in strength to interactions with water, they have been difficult to quantify. Solutes (osmolytes, denaturants) often exert large effects on these self-assembly interactions, detd. in sign and magnitude by how well the solute competes with water to interact with the relevant biopolymer surfaces. Here, an osmometric method and a water-accessible surface area (ASA) anal. are developed to quantify and interpret the interactions of the remarkable osmolyte glycine betaine (GB) with mol. surfaces in water. We find that GB, lacking hydrogen bond donors, is unable to compete with water to interact with anionic and amide oxygens; this explains its effectiveness as an osmolyte in the Escherichia coli cytoplasm. GB competes effectively with water to interact with amide and cationic nitrogens (hydrogen bonding) and esp. with arom. hydrocarbon (cation-π). The large stabilizing effect of GB on lac repressor-lac operator binding is predicted quant. from ASA information and shown to result largely from dehydration of anionic DNA phosphate oxygens in the protein-DNA interface. The incorporation of these results into theor. and computational analyses will likely improve the ability to accurately model intra- and interprotein interactions. Addnl., these results pave the way for development of solutes as kinetic/mechanistic and thermodn. probes of conformational changes and formation/disruption of mol. interfaces that occur in the steps of biomol. self-assembly processes.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1eiurjE&md5=f2b13610ab3cacbd256dbfcad93b9758
58
Torres, A. M., Grieve, S. M., Chapman, B. E., and Kuchel, P. W. (1997) Strong and weak binding of water to proteins studied by NMR triple-quantum filtered relaxation spectroscopy of (17)O-water. Biophys. Chem. 67, 187– 198, DOI: 10.1016/S0301-4622(97)00039-2
Google Scholar
There is no corresponding record for this reference.
59
Yang, P. H. and Rupley, J. A. (1979) Protein–water interactions. Heat capacity of the lysozyme–water system. Biochemistry 18, 2654– 2661, DOI: 10.1021/bi00579a035
Google Scholar
There is no corresponding record for this reference.
60
Pethig, R. (1992) Protein-water interactions determined by dielectric methods. Annu. Rev. Phys. Chem. 43, 177– 205, DOI: 10.1146/annurev.pc.43.100192.001141
Google Scholar
60
Protein-water interactions determined by dielectric methods
Pethig, R.
Annual Review of Physical Chemistry (1992), 43 (), 177-205CODEN: ARPLAP; ISSN:0066-426X.
A review with 106 refs. Dielec. theory and measurements, soln. studies, and solid-state studies are discussed.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXjsFahsA%253D%253D&md5=fffdae728e861942e5f4ce8a478c1b5c
61
Kodandapani, R., Suresh, C. G., and Vijayan, M. (1990) Crystal structure of low humidity tetragonal lysozyme at 2.1-A resolution. Variability in hydration shell and its structural consequences. J. Biol. Chem. 265, 16126– 16131
Google Scholar
There is no corresponding record for this reference.
62
Shimizu, S. and Matubayasi, N. (2014) Preferential solvation: dividing surface vs excess numbers. J. Phys. Chem. B 118, 3922– 3930, DOI: 10.1021/jp410567c
Google Scholar
62
Preferential Solvation: Dividing Surface vs Excess Numbers
Shimizu, Seishi; Matubayasi, Nobuyuki
Journal of Physical Chemistry B (2014), 118 (14), 3922-3930CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
A review. How do osmolytes affect the conformation and configuration of supramol. assembly, such as ion channel opening and actin polymn.. The key to the answer lies in the excess solvation nos. of water and osmolyte mols.; these nos. are determinable solely from exptl. data, as guaranteed by the phase rule, as we show through the exact soln. theory of Kirkwood and Buff (KB). The osmotic stress technique (OST), in contrast, purposes to yield alternative hydration nos. through the use of the dividing surface borrowed from the adsorption theory. However, we show (i) OST is equiv., when it becomes exact, to the crowding effect in which the osmolyte exclusion dominates over hydration; (ii) crowding is not the universal driving force of the osmolyte effect (e.g., actin polymn.); (iii) the dividing surface for solvation is useful only for crowding, unlike in the adsorption theory which necessitates its use due to the phase rule. KB thus clarifies the true meaning and limitations of the older perspectives on preferential solvation (such as solvent binding models, crowding, and OST), and enables excess no. detn. without any further assumptions.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXltlymtbk%253D&md5=be216ea6a9b539f63088dd901954b397
63
Zhou, H. X., Rivas, G. N., and Minton, A. P. (2008) Macromolecular crowding and confinement: Biochemical, biophysical, and potential physiological consequences. Annu. Rev. Biophys. 37, 375– 397, DOI: 10.1146/annurev.biophys.37.032807.125817
Google Scholar
63
Macromolecular crowding and confinement: Biochemical, biophysical, and potential physiological consequences
Zhou, Huan-Xiang; Rivas, German; Minton, Allen P.
Annual Review of Biophysics (2008), 37 (), 375-397CODEN: ARBNCV ISSN:. (Annual Reviews Inc.)
A review. Expected and obsd. effects of vol. exclusion on the free energy of rigid and flexible macromols. in crowded and confined systems, and consequent effects of crowding and confinement on macromol. reaction rates and equil. are summarized. Findings from relevant theor./simulation and exptl. literature published from 2004 onward are reviewed. Addnl. complexity arising from the heterogeneity of local environments in biol. media, and the presence of nonspecific interactions between macromols. over and above steric repulsion, are discussed. Theor. and exptl. approaches to the characterization of crowding- and confinement-induced effects in systems approaching the complexity of living organisms are suggested.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnsVGlurg%253D&md5=2e1fe7edb342b273b68868b02a8d137b
64
Courtenay, E. S., Capp, M. W., Anderson, C. F., and Record, M. T., Jr (2000) Vapor pressure osmometry studies of osmolyte-protein interactions: implications for the action of osmoprotectants in vivo and for the interpretation of ″osmotic stress″ experiments in vitro. Biochemistry 39, 4455– 4471, DOI: 10.1021/bi992887l
Google Scholar
64
Vapor Pressure Osmometry Studies of Osmolyte-Protein Interactions: Implications for the Action of Osmoprotectants in Vivo and for the Interpretation of "Osmotic Stress" Experiments in Vitro
Courtenay, E. S.; Capp, M. W.; Anderson, C. F.; Record, M. T., Jr.
Biochemistry (2000), 39 (15), 4455-4471CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
To interpret or predict the in vivo and in vitro responses of biopolymer processes to changes in solute concn. and coupled changes in water activity (osmotic stress), a quant. understanding of the thermodn. consequences of interactions of solutes and water with biopolymer surfaces is required. Toward this end, we report isoosmolal preferential interaction coeffs. (Γμ1) detd. by vapor pressure osmometry (VPO) over a wide range of concns. for interactions of native bovine serum albumin (BSA) with six small solutes. These solutes include Escherichia coli cytoplasmic osmolytes [potassium glutamate (K+Glu-), trehalose], E. coli osmoprotectants (proline, glycine betaine), and also glycerol and trimethylamine N-oxide (TMAO). For all six solutes, Γμ1 and the corresponding dialysis preferential interaction coeff. Γμ1,μ3 (both calcd. from the VPO data) are neg.; Γμ1,μ3 is proportional to bulk solute molality (m3bulk) at least up to 1 m (molal). Neg. values of Γμ1,μ3 indicate preferential exclusion of these solutes from a BSA soln. at dialysis equil. and correspond to local concns. of these solutes in the vicinity of BSA which are lower than their bulk concns. Of the solutes investigated, betaine is the most excluded (Γμ1,μ3/m3bulk = -49 ± 1 m-1); glycerol is the least excluded (Γμ1,μ3/m3bulk = -10 ± 1 m-1). Between these extremes, the magnitude of Γμ1,μ3/m3bulk decreases in the order glycine betaine » proline >TMAO > trehalose ≈ K+Glu- > glycerol. The order of exclusion of E. coli osmolytes from BSA surface correlates with their effectiveness as osmoprotectants, which increase the growth rate of E. coli at high external osmolality. For the most excluded solute (betaine), Γμ1,μ3 provides a min. est. of the hydration of native BSA of approx. 2.8 × 103 H2O/BSA, which corresponds to slightly less than a monolayer (estd. to be ∼3.2 × 103 H2O). Consequently, of the solutes investigated here, only betaine might be suitable for use in osmotic stress expts. in vitro as a direct probe to quantify changes in hydration of protein surface in biopolymer processes. More generally however, our results and anal. lead to the proposal that any of these solutes can be used to quantify changes in water-accessible surface area (ASA) in biopolymer processes once preferential interactions of the solute with biopolymer surface are properly taken into account.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXhvFWksL8%253D&md5=e5fa1534f3f1ba6d596387b362c98ece
65
Santoro, M. M., Liu, Y., Khan, S. M., Hou, L. X., and Bolen, D. W. (1992) Increased thermal stability of proteins in the presence of naturally occurring osmolytes. Biochemistry 31, 5278– 5283, DOI: 10.1021/bi00138a006
Google Scholar
65
Increased thermal stability of proteins in the presence of naturally occurring osmolytes
Santoro, Marcelo M.; Liu, Yufeng; Khan, Saber M. A.; Hou, Li Xiang; Bolen, D. W.
Biochemistry (1992), 31 (23), 5278-83CODEN: BICHAW; ISSN:0006-2960.
Organisms and cellular systems which have adapted to stresses such as high temp., desiccation, and urea-concg. environments have responded by concg. particular org. solutes known as osmolytes. These osmolytes are believed to confer protection to enzyme and other macromol. systems against such denaturing stresses. DSC expts. were performed on RNase A and hen egg white lysozyme in the presence of varying concns. of the osmolytes glycine, sarcosine, N,N-dimethylglycine, and betaine. Solns. contg. up to several molar concns. of these solutes resulted in considerable increases in the thermal unfolding transition temp. (Tm) for these proteins. DSC scans of RNase A in the presence of up to 8.2 M sarcosine resulted in reversible two-state unfolding transitions with Tm increases of up to 22° and unfolding enthalpy changes which were independent of Tm. On the basis of the thermodn. parameters obsd., 8.2 M sarcosine results in a stabilization free energy increase of 7.2 kcal/mol for RNase A at 65°. This translates into more than a 45,000-fold increase in stability of the native form of RNase A over that in the absence of sarcosine at this temp. Catalytic activity measurements in the presence of 4 M sarcosine give kcat and Km values that are largely unchanged from those in the absence of sarcosine. DSC of lysozyme unfolding in the presence of these osmolytes also results in Tm increases of up to 23°; however, significant irreversibility occurs with this protein. Naturally occurring glycine-based osmolytes appear to provide a general method of stabilizing proteins against thermal unfolding even well beyond the physiol. concn. range for osmolyte, and the degree of stabilization can be extraordinary.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XktVaqt70%253D&md5=fa1acd772b704d8c506795d8f7e64677
66
Tantos, A., Szrnka, K., Szabo, B., Bokor, M., Kamasa, P., Matus, P., Bekesi, A., Tompa, K., Han, K. H., and Tompa, P. (2013) Structural disorder and local order of hNopp140. Biochim. Biophys. Acta, Proteins Proteomics 1834, 342– 350, DOI: 10.1016/j.bbapap.2012.08.005
Google Scholar
66
Structural disorder and local order of hNopp140
Tantos, Agnes; Szrnka, Kriszta; Szabo, Beata; Bokor, Monika; Kamasa, Pawel; Matus, Peter; Bekesi, Angela; Tompa, Kalman; Han, Kyou-Hoon; Tompa, Peter
Biochimica et Biophysica Acta, Proteins and Proteomics (2013), 1834 (1), 342-350CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)
Human nucleolar phosphoprotein p140 (hNopp 140) is a highly phosphorylated protein inhibitor of casein kinase 2 (CK2). As in the case of many kinase-inhibitor systems, the inhibitor has been described to belong to the family of intrinsically disordered proteins (IDPs), which often utilize transient structural elements to bind their cognate enzyme. Here, the authors investigated the structural status of this protein both to provide distinct lines of evidence for its disorder and to point out its transient structure potentially involved in interactions and also its tendency to aggregate. The structural disorder of hNopp140 was apparent by its anomalous electrophoretic mobility, protease sensitivity, heat stability, hydrodynamic behavior on size-exclusion chromatog., 1H NMR spectrum, and DSC calorimetry scan. Phosphoprotein hNopp140 had a significant tendency to aggregate and the change of its CD spectrum in the presence of 0-80% trifluoroethanol suggested a tendency to form local helical structures. Wide-line NMR measurements suggested the overall disordered character of the protein. In all, the data suggested that this protein falls into the pre-molten globule state of IDPs, with a significant tendency to become ordered in the presence of its partner as demonstrated in the presence of transcription factor IIB (TFIIB).
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsV2qtLbI&md5=a69400cb1225796b669940c07d12fc11
67
van der Lee, R., Buljan, M., Lang, B., Weatheritt, R. J., Daughdrill, G. W., Dunker, A. K., Fuxreiter, M., Gough, J., Gsponer, J., Jones, D. T., Kim, P. M., Kriwacki, R. W., Oldfield, C. J., Pappu, R. V., Tompa, P., Uversky, V. N., Wright, P. E., and Babu, M. M. (2014) Classification of intrinsically disordered regions and proteins. Chem. Rev. 114, 6589– 6631, DOI: 10.1021/cr400525m
Google Scholar
67
Classification of intrinsically disordered regions and proteins
van der Lee, Robin; Buljan, Marija; Lang, Benjamin; Weatheritt, Robert J.; Daughdrill, Gary W.; Dunker, A. Keith; Fuxreiter, Monika; Gough, Julian; Gsponer, Joerg; Jones, David T.; Kim, Philip M.; Kriwacki, Richard W.; Oldfield, Christopher J.; Pappu, Rohit V.; Tompa, Peter; Uversky, Vladimir N.; Wright, Peter E.; Babu, M. Madan
Chemical Reviews (Washington, DC, United States) (2014), 114 (13), 6589-6631CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
A review. While many proteins need to adopt a well-defined structure to carry out their function, a large fraction of the proteome of any organism consists of polypeptide segments that are not likely to form a defined 3-dimensional structure, but are nevertheless functional. These protein segments are referred to as intrinsically disordered regions (IDRs). Proteins without IDRs are called structured proteins, and proteins with entirely disordered sequences that do not adopt any tertiary structure are referred to as intrinsically disordered proteins (IDPs). IDRs and IDPs are prevalent in eukaryotic genomes, with 44% of human protein-coding genes contg. disordered segments of >30 residues in length. Here, the authors discuss classification approaches based on function, functional elements, sequence, protein interactions, evolution, regulation, and biophys. properties. The authors also discuss resources that are currently available for gaining insight into IDR function, suggest areas where increased efforts are likely to advance the understanding of the functions of protein disorder, and speculate how combinations of multiple existing classification schemes could achieve high quality function prediction for IDRs, which should ultimately lead to improved function coverage and a deeper understanding of protein function.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXntFChur0%253D&md5=436cf71d9b77f6f3b176233a6abd9628
68
Uversky, V. N. (2013) The most important thing is the tail: multitudinous functionalities of intrinsically disordered protein termini. FEBS Lett. 587, 1891– 1901, DOI: 10.1016/j.febslet.2013.04.042
Google Scholar
There is no corresponding record for this reference.
69
Uversky, V. N. (2009) Intrinsically disordered proteins and their environment: effects of strong denaturants, temperature, pH, counter ions, membranes, binding partners, osmolytes, and macromolecular crowding. Protein J. 28, 305– 325, DOI: 10.1007/s10930-009-9201-4
Google Scholar
69
Intrinsically disordered proteins and their environment: Effects of strong denaturants, temperature, pH, counter ions, membranes, binding partners, osmolytes, and macromolecular crowding
Uversky, Vladimir N.
Protein Journal (2009), 28 (7-8), 305-325CODEN: PJROAH; ISSN:1572-3887. (Springer)
Av review. Intrinsically disordered proteins (IDPs) differ from "normal" ordered proteins at several levels, structural, functional and conformational. Amino acid biases characteristic for IDPs det. their structural variability and lack of rigid well-folded structure. This structural plasticity is necessary for the unique functional repertoire of IDPs, which is complementary to the catalytic activities of ordered proteins. Amino acid biases also drive atypical responses of IDPs to changes in their environment. The conformational behavior of IDPs is characterized by the low cooperativity (or the complete lack thereof) of denaturant-induced unfolding, lack of the measurable excess heat absorption peak(s) characteristic for the melting of ordered proteins, "turned out" response to heat and changes in pH, the ability to gain structure in the presence of various counter ions, osmolytes, membranes, and binding partners, and by the unique response to macromol. crowding. Here, the author describes some of the most characteristic features of IDP conformational behavior and the unique response of IDPs to changes in their environment.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtFGjt7jE&md5=84a0c51d56bf800bd65f0e3318a8c7d7
70
Abeln, S. and Frenkel, D. (2008) Disordered flanks prevent peptide aggregation. PLoS Comput. Biol. 4, e1000241 DOI: 10.1371/journal.pcbi.1000241
Google Scholar
There is no corresponding record for this reference.
71
Grana-Montes, R., Marinelli, P., Reverter, D., and Ventura, S. (2014) N-terminal protein tails act as aggregation protective entropic bristles: the SUMO case. Biomacromolecules 15, 1194– 1203, DOI: 10.1021/bm401776z
Google Scholar
71
N-Terminal Protein Tails Act as Aggregation Protective Entropic Bristles: The SUMO Case
Grana-Montes, Ricardo; Marinelli, Patrizia; Reverter, David; Ventura, Salvador
Biomacromolecules (2014), 15 (4), 1194-1203CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
The formation of β-sheet enriched amyloid fibrils constitutes the hallmark of many diseases but is also an intrinsic property of polypeptide chains in general, because the formation of compact globular proteins comes at the expense of an inherent sequential aggregation propensity. In this context, identification of strategies that enable proteins to remain functional and sol. in the cell has become a central issue in chem. biol. We show here, using human SUMO proteins as a model system, that the recurrent presence of disordered tails flanking globular domains might constitute yet another of these protective strategies. These short, disordered, and highly sol. protein segments would act as intramol. entropic bristles, reducing the overall protein intrinsic aggregation propensity and favoring thus the attainment and maintenance of functional conformations.
>> More from SciFinder ^®
https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXivFyqurs%253D&md5=d77448eec5800f23bda5fd4c25a812fb
72
Strader, M. B. (1998) Constructing a hybrid of R67 dihydrofolate reductase to study asymmetric mutations in the active site. M.S. Thesis, University of Tennessee, Knoxville, TN.
Google Scholar
There is no corresponding record for this reference.
73
Martinez, M. A., Pezo, V., Marliere, P., and Wain-Hobson, S. (1996) Exploring the functional robustness of an enzyme by in vitro evolution. EMBO J. 15, 1203– 1210
Google Scholar
There is no corresponding record for this reference.

Cited By

Click to copy section linkSection link copied!

This article is cited by 6 publications.

Michael R. Duff, Jr., Nidhi Desai, Michael A. Craig, Pratul K. Agarwal, Elizabeth E. Howell. Crowders Steal Dihydrofolate Reductase Ligands through Quinary Interactions. Biochemistry 2019, 58 (9) , 1198-1213. https://doi.org/10.1021/acs.biochem.8b01110
Claudèle Lemay-St-Denis, Joelle N. Pelletier. From a binding module to essential catalytic activity: how nature stumbled on a good thing. Chemical Communications 2023, 59 (84) , 12560-12572. https://doi.org/10.1039/D3CC04209J
Chen Yu, Dong Liang, Cui Yang, Bing Zhao, Qin Dong, Aoli Wu, Lian Li, Hengchang Zang. Research progress and the application of near-infrared spectroscopy in protein structure and molecular interaction analysis. Vibrational Spectroscopy 2022, 121 , 103390. https://doi.org/10.1016/j.vibspec.2022.103390
Claudèle Lemay-St-Denis, Sarah-Slim Diwan, Joelle N. Pelletier. The Bacterial Genomic Context of Highly Trimethoprim-Resistant DfrB Dihydrofolate Reductases Highlights an Emerging Threat to Public Health. Antibiotics 2021, 10 (4) , 433. https://doi.org/10.3390/antibiotics10040433
Gabriel J. Fuente‐Gómez, Creighton L. Kellum, Alexis C. Miranda, Michael R. Duff, Elizabeth E. Howell. Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19 F NMR . Protein Science 2021, 30 (2) , 477-484. https://doi.org/10.1002/pro.4007
Dušan Petrović, Valeria A. Risso, Shina Caroline Lynn Kamerlin, Jose M. Sanchez-Ruiz. Conformational dynamics and enzyme evolution. Journal of The Royal Society Interface 2018, 15 (144) , 20180330. https://doi.org/10.1098/rsif.2018.0330

Download PDF

Get e-Alerts

Biochemistry

Cite this: Biochemistry 2017, 56, 44, 5886–5899

Click to copy citationCitation copied!

https://doi.org/10.1021/acs.biochem.7b00822

Published October 11, 2017

Request reuse permissions

Article Views

Altmetric

Citations

Learn about these metrics

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

Abstract
High Resolution Image
Download MS PowerPoint Slide
Figure 1
Figure 1. SANS profile and GNOM analysis for apo, full-length R67 DHFR. (A) Normalized scattering intensity of the protein, I(q)/I(0), with an increase in q. The SANS profile was obtained by subtracting the scattering contribution from the buffer and normalizing the scattering intensity by I(0). (B) A GNOM fit of the profile gave the pairwise distance distribution and an R_g value of 21.89 ± 0.12 Å.
High Resolution Image
Download MS PowerPoint Slide
Figure 2
Figure 2. SASSIE analysis of apo R67 DHFR suggests compaction of the N-termini. (A) Analysis of the frames generated by directed Monte Carlo sampling (117000, black filled circles) and frames from the MD runs (154000, cyan filled circles). The red line indicates a χ² = 10 cutoff. Data with χ² > 50 are not shown for the sake of clarity. (B) Overlays of the theoretical SANS profiles for the best [χ² = 1.8 (red squares)] and worst [χ² = 430 (blue triangles)] fits compared to the experimental SANS data (black circles). (C) Corresponding best and worst structures. (D) Density plot describing all the space sampled by MD and Monte Carlo structures (dark gray mesh) representing most of the available “structural space”. The structures identified by SASSIE as providing good fits to the experimental SANS data are shown by green mesh. One of the good models describing full-length homotetrameric R67 DHFR is shown in the center of the mesh.
High Resolution Image
Download MS PowerPoint Slide
Figure 3
Figure 3. SASSIE analysis of ligand-bound R67 DHFR complexes indicates compaction of the N-termini. (A) GNOM fits of the pairwise distribution plots of R_g for apo R67 DHFR (green line), R67 DHFR–NADP⁺ binary (dashed orange line), and R67 DHFR–NADP⁺–DHF ternary (dotted blue line) complexes. SANS data were collected using 6.05 mg/mL DHFR in 20 mM deuterated Tris buffer in D₂O (pD 7.0) with no osmolyte. Binary or ternary complexes were formed by adding 3 mM NADP⁺ or NADP⁺ with 2 mM DHF, respectively. The R_g values for the apoprotein, binary complex, and ternary complex are 21.89 ± 0.12, 21.45 ± 0.14, and 21.45 ± 0.18 Å, respectively. While it may seem that the maximal diameter of the protein (D_max) varies, it is more that there is not a well-defined D_max value for proteins with flexible regions. Rather, a small range of D_max values all appear satisfactory. Within this “optimized” range, R_g and I(0) values are not changing significantly, allowing reliable parameters to be gained from the analysis. (B) Comparison of the mesh plots obtained from SASSIE for the apo (green) and NADP⁺–DHF ternary (blue) complexes. Bound DHF (cyan) and NADPH (magenta) are shown as ball-and-stick models in the center of the active site pore. (C) Venn diagram comparing the overlaps associated with the number of apo, binary, and ternary best fits. Figure S6 shows similar figures for the binary complex.
High Resolution Image
Download MS PowerPoint Slide
Figure 4
Figure 4. SASSIE analysis of apo R67 DHFR in the presence of 20% deuterated betaine indicates compact as well as partially extended N-terminal conformations. (A) GNOM fits for the pairwise distribution of R_g for apo R67 DHFR with and without 20% deuterated betaine. SANS data were collected for R67 DHFR at 6.5 mg/mL in 20 mM deuterated Tris buffer in D₂O (pD 7.0) with 20% deuterated betaine (dashed purple line). A wider distribution in the presence of betaine and an R_g value of 23.0 ± 0.30 Å indicate an increased number of slightly more extended conformations for the termini of R67 DHFR. (B) Overlay of the density plots for the best frames with χ² values of <10 for the deuterated betaine protein sample (purple mesh) and for the apoprotein without betaine (green mesh).
High Resolution Image
Download MS PowerPoint Slide
Figure 5
Figure 5. Preferential hydration of the full-length and truncated R67 DHFR in the presence of osmolytes. Small angle neutron scattering intensity ratios with and without osmolyte [I(0)_s/I(0)] as a function of osmolyte concentration, f_v, for betaine (w/v) (magenta filled squares) and DMSO (v/v) (green filled triangles). Panels A and B show the data for full-length and truncated R67 DHFRs, respectively. Solid lines are fits to eq 2 to calculate the number of protein-associated waters, n_w.
High Resolution Image
Download MS PowerPoint Slide
Figure 6
Figure 6. (A) Effects of osmolytes on thermal denaturation of full-length R67 DHFR. DSC scans were performed with 150–160 μM R67 DHFR in MTA buffer with and without 20% betaine (magenta) and 15% DMSO (green). Betaine increases the melting temperature of the protein by 2–3 °C, whereas DMSO decreases it by 7–9 °C. (B) DSC thermograms for truncated R67 (50–90 μM) DHFR in MTA buffer, pH 8.0 buffer (black line), or buffer with 20% betaine (red) or 15% DMSO (cyan). Panels C and D show our PPC results. Thermal expansion coefficients (α_s) were obtained from PPC using 2.5 °C increments between 10 and 95 °C for (C) full-length R67 DHFR (90–150 μM) or (D) truncated R67 DHFR (99–150 μM). Experiments were performed in 45 mM Na₂HPO₄, pH 8.0 buffer alone (●), buffer with 10% betaine (○), or buffer with 20% betaine (△).
High Resolution Image
Download MS PowerPoint Slide
Figure 7
Figure 7. Surface of R67 DHFR (2RH2) shown with the exposed lipophilic surfaces colored green and hydrophilic surfaces colored magenta along the monomer–monomer interface. The surface was made partially transparent so the side chains could be seen. Two symmetry-related W45 residues are labeled in the center of the figure. This figure was composed using the lipophilic surface option in MOE (version 2015.10).
High Resolution Image
Download MS PowerPoint Slide
References
This article references 73 other publications.
1. 1
  Toulouse, J. L., Edens, T. J., Alejaldre, L., Manges, A. R., and Pelletier, J. N. (2017) Integron-Associated DfrB4, a Previously Uncharacterized Member of the Trimethoprim-Resistant Dihydrofolate Reductase B Family, Is a Clinically Identified Emergent Source of Antibiotic Resistance. Antimicrob. Agents Chemother. 61, e02665-16, DOI: 10.1128/AAC.02665-16
  There is no corresponding record for this reference.
2. 2
  Bastien, D., Ebert, M. C., Forge, D., Toulouse, J., Kadnikova, N., Perron, F., Mayence, A., Huang, T. L., Vanden Eynde, J. J., and Pelletier, J. N. (2012) Fragment-based design of symmetrical bis-benzimidazoles as selective inhibitors of the trimethoprim-resistant, type II R67 dihydrofolate reductase. J. Med. Chem. 55, 3182– 3192, DOI: 10.1021/jm201645r
  2
  Fragment-Based Design of Symmetrical Bis-benzimidazoles as Selective Inhibitors of the Trimethoprim-Resistant, Type II R67 Dihydrofolate Reductase
  Bastien, Dominic; Ebert, Maximilian C. C. J. C.; Forge, Delphine; Toulouse, Jacynthe; Kadnikova, Natalia; Perron, Florent; Mayence, Annie; Huang, Tien L.; Vanden Eynde, Jean Jacques; Pelletier, Joelle N.
  Journal of Medicinal Chemistry (2012), 55 (7), 3182-3192CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
  The continuously increasing use of trimethoprim as a common antibiotic for medical use and for prophylactic application in terrestrial and aquatic animal farming has increased its prevalence in the environment. This has been accompanied by increased drug resistance, generally in the form of alterations in the drug target, dihydrofolate reductase (DHFR). The most highly resistant variants of DHFR are known as type II DHFR, among which R67 DHFR is the most broadly studied variant. We report the first attempt at designing specific inhibitors to this emerging drug target by fragment-based design. The detection of inhibition in R67 DHFR was accompanied by parallel monitoring of the human DHFR, as an assessment of compd. selectivity. By those means, small arom. mols. of 150-250 g/mol (fragments) inhibiting R67 DHFR selectively in the low millimolar range were identified. More complex, sym. bis-benzimidazoles and a bis-carboxyphenyl were then assayed as fragment-based leads, which procured selective inhibition of the target in the low micromolar range (Ki = 2-4 μM). The putative mode of inhibition is discussed according to mol. modeling supported by in vitro tests.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XktVynsLo%253D&md5=e648c46e90ea8930a85a0b4886b468c2
3. 3
  Toulouse, J. L., Abraham, S. M. J., Kadnikova, N., Bastien, D., Gauchot, V., Schmitzer, A. R., and Pelletier, J. N. (2017) Investigation of Classical Organic and Ionic Liquid Cosolvents for Early-Stage Screening in Fragment-Based Inhibitor Design with Unrelated Bacterial and Human Dihydrofolate Reductases. Assay Drug Dev. Technol. 15, 141– 153, DOI: 10.1089/adt.2016.768
  3
  Investigation of Classical Organic and Ionic Liquid Cosolvents for Early-Stage Screening in Fragment-Based Inhibitor Design with Unrelated Bacterial and Human Dihydrofolate Reductases
  Toulouse, Jacynthe L.; Abraham, Sarah M. J.; Kadnikova, Natalia; Bastien, Dominic; Gauchot, Vincent; Schmitzer, Andreea R.; Pelletier, Joelle N.
  Assay and Drug Development Technologies (2017), 15 (4), 141-153CODEN: ADDTAR; ISSN:1540-658X. (Mary Ann Liebert, Inc.)
  Drug design by methods such as fragment screening requires effective solubilization of millimolar concns. of small org. compds. while maintaining the properties of the biol. target. We investigate four org. solvents and three 1-butyl-3-methylimidazolium (BMIm)-based ionic liqs. (ILs) as cosolvents to establish conditions for screening two structurally unrelated dihydrofolate reductases (DHFRs) that are prime drug targets. Moderate concns. (10%-15%) of cosolvents had little effect on inhibition of the microbial type II R67 DHFR and of human DHFR (hDHFR), while higher concns. of org. cosolvents generally decreased activity of both DHFRs. In contrast, a specific IL conserved the activity of one DHFR, while severely reducing the activity of the other, and vice versa, illustrating the differing effect of ILs on distinct protein folds. Most of the cosolvents investigated preserved the fold of R67 DHFR and had little effect on binding of the cofactor NADPH, but reduced the productive affinity for its substrate. In contrast, cosolvents resulted in modest structural destabilization of hDHFR with little effect on productive affinity. We conclude that the org. cosolvents, methanol, DMF, and dimethylsulfoxide, offer the most balanced conditions for early-stage compd. screening as they maintain sufficient biol. activity of both DHFRs while allowing for compd. dissoln. in the millimolar range. However, IL cosolvents showed poor capacity to solubilize org. compds. at millimolar concns., mitigating their utility in early-stage screening. Nonetheless, ILs could provide an alternative to classical org. cosolvents when low concns. of inhibitors are used, as when characterizing higher affinity inhibitors.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVantrfO&md5=0498186b3b64f5d33a9473b9c5bca87a
4. 4
  Narayana, N., Matthews, D. A., Howell, E. E., and Xuong, N. (1995) A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site,. Nat. Struct. Mol. Biol. 2, 1018– 1025, DOI: 10.1038/nsb1195-1018
  4
  A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site
  Narayana, Narendra; Matthews, David A.; Howell, Elizabeth E.; Xuong, Nguyen-huu
  Nature Structural Biology (1995), 2 (11), 1018-25CODEN: NSBIEW; ISSN:1072-8368. (Nature Publishing Co.)
  Bacteria expressing R67-plasmid encoded dihydrofolate reductase (R67 DHFR) exhibited high-level resistance to the antibiotic trimethoprim. Native R67 DHFR is a 34,000 Mr homotetramer which exists in equil. with an inactive dimeric form. The structure of native R67 DHFR has now been solved at 1.7 Å resoln. and is unrelated to that of chromosomal DHFR. Homotetrameric R67 DHFR has an unusual pore, 25 Å in length, passing through the middle of the mol. Two folate mols. bind asym. within the pore indicating that the enzyme's active site consists of symmetry related binding surfaces from all four identical units.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXptlegtrc%253D&md5=23ff4e76931aeef868529123d7f91a02
5. 5
  Feng, J., Grubbs, J., Dave, A., Goswami, S., Horner, C. G., and Howell, E. E. (2010) Radical redesign of a tandem array of four R67 dihydrofolate reductase genes yields a functional, folded protein possessing 45 substitutions. Biochemistry 49, 7384– 7392, DOI: 10.1021/bi1005943
  5
  Radical Redesign of a Tandem Array of Four R67 Dihydrofolate Reductase Genes Yields a Functional, Folded Protein Possessing 45 Substitutions
  Feng, Jian; Grubbs, Jordan; Dave, Ashita; Goswami, Sumit; Horner, Caroline Glyn; Howell, Elizabeth E.
  Biochemistry (2010), 49 (34), 7384-7392CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  R67 dihydrofolate reductase (DHFR) is a plasmid-encoded, type II enzyme. Four monomers (78 amino acids long) assemble into a homotetramer possessing 222 symmetry. In previous studies, a tandem array of four R67 DHFR gene copies was fused in frame to generate a functional monomer named Quad1. This protein possessed the essential tertiary structure of the R67 "parent". To facilitate mutagenesis reactions, restriction enzyme sites were introduced in the tandem gene array. S59A and H362L mutations were also added to minimize possible folding topologies; this protein product, named Quad3, possesses 10 substitutions and is functional. Since R67 DHFR possesses a stable scaffold, a large jump in sequence space was performed by the further addn. of 45 amino acid substitutions. The mutational design utilized alternate sequences from other type II DHFRs. In addn., most of the mutations were positioned on the surface of the protein as well as in the disordered N-terminal sequence, which serves as the linker between the fused domains. The resulting Quad4 protein is quite functional; however, it is less stable than Quad1, suffering a ΔΔG loss of 5 kcal/mol at pH 5. One unexpected result was formation of Quad4 dimers and higher order oligomers at pH 8. R67 DHFR, and its deriv. Quad proteins, possesses a robust scaffold, capable of withstanding introduction of ≥55 substitutions.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXpslKjs7s%253D&md5=635f32ee7e52a6c1325ee7f76c8a65ca
6. 6
  Matthews, D. A., Smith, S. L., Baccanari, D. P., Burchall, J. J., Oatley, S. J., and Kraut, J. (1986) Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67. Biochemistry 25, 4194– 4204, DOI: 10.1021/bi00363a005
  6
  Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67
  Matthews, David A.; Smith, S. L.; Baccanari, D. P.; Burchall, J. J.; Oatley, S. J.; Kraut, J.
  Biochemistry (1986), 25 (15), 4194-204CODEN: BICHAW; ISSN:0006-2960.
  Cryst. R67 dihydrofolate reductase (DHFR) is a dimeric mol. with 2 identical 78-amino-acid-residue subunits, each folded into a β-barrel conformation. The outer surfaces of the 3 longest β-strands in each protomer together form a 3rd β-barrel having 6 strands at the subunit interface. A unique feature of the enzyme structure is that while the intersubunit β-barrel is quite regular over most of its surface, an 8-Å gap runs the full length of the barrel, disrupting potential H bonds between β-strand D in subunit I and the adjacent corresponding strand of subunit II. It is proposed that this deep groove is the NADPH-binding site and that the assocn. between protein and cofactor is modulated by H-bonding interactions along one face of this antiparallel β-barrel structure. A hypothetical model is proposed for the R67 DHFR-NADPH-folate ternary complex that is consistent with both the known reaction stereoselectivity and the weak binding of 2,4-diamino inhibitors to the plasmid-specified reductase. Geometrical comparison of this model with an exptl. detd. structure for chicken DHFR suggests that chromosomal and type II R-plasmid-specified enzymes may have independently evolved similar catalytic machinery for substrate redn.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XksFKqtrk%253D&md5=00f5fc4a2ebdeea18be9e89909f7d2c7
7. 7
  Reece, L. J., Nichols, R., Ogden, R. C., and Howell, E. E. (1991) Construction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein. Biochemistry 30, 10895– 10904, DOI: 10.1021/bi00109a013
  7
  Construction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein
  Reece, Lisa J.; Nichols, Robert; Ogden, Richard C.; Howell, Elizabeth E.
  Biochemistry (1991), 30 (45), 10895-904CODEN: BICHAW; ISSN:0006-2960.
  Plasmid R67 dihydrofolate reductase (DHFR) is a novel protein that provides clin. resistance to the antibacterial drug, trimethoprim. The previously published crystal structure of a dimeric form of R67 DHFR indicated that the 1st 16 amino acids are disordered. To investigate whether these amino acids are necessary for protein function, the 1st 16 N-terminal residues were cleaved off by chymotrypsin. The truncated protein was found to be fully active with kcat = 1.3 s-1, Km(NADPH) = 3.0 μM, and Km(dihydrofolate) = 5.8 μM. This result suggests that the functional core of the protein resides in the β-barrel structure defined by residues 27-78. To study this protein further, synthetic genes coding for full-length and truncated R67 DHFRs were constructed. Surprisingly, the gene coding for truncated R67 DHFR did not produce protein in vivo or confer trimethoprim resistance upon Escherichia coli. Therefore, the relative stabilities of native and truncated R67 DHFR were investigated by equil. unfolding studies. The unfolding of dimeric native R67 DHFR was protein concn.-dependent and could be described by a 2-state model involving native dimer and unfolded monomer. Using absorbance, fluorescence, and CD techniques, an av. ΔGH2O of 13.9 kcal/mol was found for native R67 DHFR. In contrast, an av.ΔGH2O of 11.3 kcal/mol was obsd. for truncated R67 DHFR. These results indicated that native R67 DHFR is 2.6 kcal/mol more stable than the truncated protein. This stability difference may be part of the reason why protein from the truncated gene is not found in vivo in E. coli.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXmslCntbc%253D&md5=1191aeea031ad497bff25437b10c4d57
8. 8
  Krahn, J. M., Jackson, M. R., DeRose, E. F., Howell, E. E., and London, R. E. (2007) Crystal structure of a type II dihydrofolate reductase catalytic ternary complex. Biochemistry 46, 14878– 14888, DOI: 10.1021/bi701532r
  8
  Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex
  Krahn, Joseph M.; Jackson, Michael R.; DeRose, Eugene F.; Howell, Elizabeth E.; London, Robert E.
  Biochemistry (2007), 46 (51), 14878-14888CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a sym. homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR·DHF·NADP+ catalytic complex, resolved to 1.26 Å. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine ring. This set of interactions orients the arom. rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Addnl., a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67'-Y69' residues provides an unusually tight interface, which appears to serve as a "mol. clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addn. to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationally challenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlOnur3O&md5=68e1db370224ac27699b3a46cdf5c49c
9. 9
  Narayana, N. (2006) High-resolution structure of a plasmid-encoded dihydrofolate reductase: pentagonal network of water molecules in the D2-symmetric active site. Acta Crystallogr., Sect. D: Biol. Crystallogr. 62, 695– 706, DOI: 10.1107/S0907444906014764
  There is no corresponding record for this reference.
10. 10
  Amyes, S. G. and Smith, J. T. (1976) The purification and properties of the trimethoprim-resistant dihydrofolate reductase mediated by the R-factor, R388. Eur. J. Biochem. 61, 597– 603, DOI: 10.1111/j.1432-1033.1976.tb10055.x
  10
  The purification and properties of the trimethoprim-resistant dihydrofolate reductase mediated by the R-factor, R388
  Amyes, Sebastian G. B.; Smith, John T.
  European Journal of Biochemistry (1976), 61 (2), 597-603CODEN: EJBCAI; ISSN:0014-2956.
  The R-factor, R 388, mediates the prodn. of a trimethoprim-resistant dihydrofolate reductase. This enzyme has a different mol. wt. and pH profile than the trimethoprim-sensitive enzyme of the Escherichia coli host. The R-factor mediated enzyme was sepd. completely from the host E. coli enzyme by DEAE-cellulose ion-exchange chromatog. The purified R-factor enzyme was ∼20,000 times less susceptible to trimethoprim than the E. coli enzyme and although it was inhibited competitively by trimethoprim, its Ki was 20,000 times greater than that of the host enzyme. The R388 and E. coli enzymes also differed in their substrate specificity requirements. In addn., the R388 enzyme surprisingly conferred high level resistance to the broad spectrum dihydrofolate reductase inhibitor, amethopterin. The possible origins of the R388 enzyme are discussed.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XhtVWgtr0%253D&md5=431d7961c3c4d8ebf230b6f2d31c66ae
11. 11
  Flensburg, J. and Steen, R. (1986) Nucleotide sequence analysis of the trimethoprim resistant dihydrofolate reductase encoded by R plasmid R751. Nucleic Acids Res. 14, 5933, DOI: 10.1093/nar/14.14.5933
  There is no corresponding record for this reference.
12. 12
  Alonso, H. and Gready, J. E. (2006) Integron-sequestered dihydrofolate reductase: a recently redeployed enzyme. Trends Microbiol. 14, 236– 242, DOI: 10.1016/j.tim.2006.03.003
  12
  Integron-sequestered dihydrofolate reductase: A recently redeployed enzyme
  Alonso, Hernan; Gready, Jill E.
  Trends in Microbiology (2006), 14 (5), 236-242CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)
  A review. The introduction and wide use of antibacterial drugs has resulted in the emergence of resistant organisms. DfrB dihydrofolate reductase (DHFR) is a bacterial enzyme that is uniquely assocd. with mobile gene cassettes within integrons, and confers resistance to the drug trimethoprim. This enzyme has intrigued microbiologists since it was discovered more than thirty years ago because of its simple structure, enzymic inefficiency and its virtual insensitivity to trimethoprim. Here, for the first time, a comprehensive discussion of genetic, evolutionary, structural and functional studies of this enzyme is presented together. This information supports the ideas that DfrB DHFR is a poorly adapted catalyst and has recently been recruited to perform a novel enzymic activity in response to selective pressure.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xks1ektL0%253D&md5=0fedb66b8aabc150deee62184e4ab725
13. 13
  Strader, M. B. (2003) Identifying the Catalytic and Ligand Binding Roles of Active Site Residues in Homotetrameric R67 Dihydrofolate Reductase. Ph.D. Dissertation, University of Tennessee, Knoxville, TN.
  There is no corresponding record for this reference.
14. 14
  Duff, M. R., Jr., Chopra, S., Strader, M. B., Agarwal, P. K., and Howell, E. E. (2016) Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase. Biochemistry 55, 133– 145, DOI: 10.1021/acs.biochem.5b00981
  14
  Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase
  Duff, Michael R.; Chopra, Shaileja; Strader, Michael Brad; Agarwal, Pratul K.; Howell, Elizabeth E.
  Biochemistry (2016), 55 (1), 133-145CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by NMR and crystallog., is disordered when bound. To explore whether smaller active site vols. (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asym. mutations that decreased the half-pore vol. by ∼35% were constructed. Only minor effects on kcat were obsd. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated crosslinking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that crosslinking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both neg. charged carboxylates in the glutamate tail were replaced with pos. charged amines. The Ki for this adduct was ∼9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFCqsbbL&md5=57afbe6aebb421eab08bf43e8b083ef5
15. 15
  Schmitzer, A. R., Lepine, F., and Pelletier, J. N. (2004) Combinatorial exploration of the catalytic site of a drug-resistant dihydrofolate reductase: creating alternative functional configurations. Protein Eng., Des. Sel. 17, 809– 819, DOI: 10.1093/protein/gzh090
  15
  Combinatorial exploration of the catalytic site of a drug-resistant dihydrofolate reductase: creating alternative functional configurations
  Schmitzer, Andreea R.; Lepine, Francois; Pelletier, Joelle N.
  Protein Engineering, Design & Selection (2004), 17 (11), 809-819CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)
  We have applied a global approach to enzyme active site exploration, where multiple mutations were introduced combinatorially at the active site of Type II R67 dihydrofolate reductase (R67 DHFR), creating numerous new active site environments within a const. framework. By this approach, we combinatorially modified all 16 principal amino acids that constitute the active site of this enzyme. This approach is fundamentally different from active site point mutation in that the native active site context is no longer accounted for. Among the 1536 combinatorially mutated active site variants of R67 DHFR we created, we selected and kinetically characterized three variants with highly altered active site compns. We detd. that they are of high fitness, as defined by a complex function consisting jointly of catalytic activity and resistance to trimethoprim. The kcat and KM values were similar to those for the native enzyme. The favorable Δ(ΔG) values obtained (ranging from -0.72 to -1.08 kcal/mol) suggest that, despite their complex mutational pattern, no fundamental change in the catalytic mechanism has occurred. We illustrate that combinatorial active site mutagenesis can allow for the creation of compensatory mutations that could not be predicted and thus provides a route for more extensive exploration of functional sequence space than is allowed by point mutation.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhsFSntb8%253D&md5=3563570b33610340fa17e16761e3591a
16. 16
  Bradrick, T. D., Shattuck, C., Strader, M. B., Wicker, C., Eisenstein, E., and Howell, E. E. (1996) Redesigning the quaternary structure of R67 dihydrofolate reductase. Creation of an active monomer from a tetrameric protein by quadruplication of the gene. J. Biol. Chem. 271, 28031– 28037, DOI: 10.1074/jbc.271.45.28031
  There is no corresponding record for this reference.
17. 17
  Dam, J. and Blondel, A. (2004) Effect of multiple symmetries on the association of R67 DHFR subunits bearing interfacial complementing mutations. Protein Sci. 13, 1– 14, DOI: 10.1110/ps.03309504
  17
  Effect of multiple symmetries on the association of R67 DHFR subunits bearing interfacial complementing mutations
  Dam, Julie; Blondel, Arnaud
  Protein Science (2004), 13 (1), 1-14CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
  It was shown previously that complementation could be a powerful mean to probe protein-protein interactions in the normally tetrameric R67 DHFR. Indeed, mixing complementing inactive dimeric mutants produced active heterotetramers. This approach turned a homo-oligomer into a hetero-oligomer and thus allowed the use of combinatorial assays, a subtle anal. of the assocn. forces, and a precise detn. of the equil. dissocn. consts. (KD) by titrimetry. However, for some of the complementing pairs, the exptl. data implied multiple equil. involving heterodimers, although no monomers could be detected. Thus, the reactions involved had to be identified to elaborate a suitable model to det. the KD of those pairs correctly. That model suggested that homodimers assocd. rapidly before the protomers could be redistributed in a multiple equil. system. Kinetic data confirmed that view. The assocn. data at equil. were analyzed by multiple curve fitting with all plausible combinations of parameters. This gave a confidence interval for KD that is safer than the usual 67% or 90% confidence interval. Finally, the KD of one specific reaction, the dissocn. of a heterotetramer with the relevant symmetry into two homodimers could be detd. with the relevant model for each complementing pair, although multiple equil. were present. These KD can thus be used as a set of refs. data to test and improve theor. methods such as assocn. free energy calcns.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhsFWjsg%253D%253D&md5=914326e8e1a24f83662e543b2e10df86
18. 18
  Dam, J., Rose, T., Goldberg, M. E., and Blondel, A. (2000) Complementation between dimeric mutants as a probe of dimer-dimer interactions in tetrameric dihydrofolate reductase encoded by R67 plasmid of E. coli. J. Mol. Biol. 302, 235– 250, DOI: 10.1006/jmbi.2000.4051
  There is no corresponding record for this reference.
19. 19
  Yachnin, B. J., Colin, D. Y., Volpato, J. P., Ebert, M., Pelletier, J. N., and Berghuis, A. M. (2011) Novel crystallization conditions for tandem variant R67 DHFR yield a wild-type crystal structure. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 67, 1316– 1322, DOI: 10.1107/S1744309111030417
  19
  Novel crystallization conditions for tandem variant R67 DHFR yield a wild-type crystal structure
  Yachnin, Brahm J.; Colin, Damien Y.; Volpato, Jordan P.; Ebert, Maximilian; Pelletier, Joelle N.; Berghuis, Albert M.
  Acta Crystallographica, Section F: Structural Biology and Crystallization Communications (2011), 67 (11), 1316-1322CODEN: ACSFCL; ISSN:1744-3091. (International Union of Crystallography)
  Trimethoprim is an antibiotic that targets bacterial dihydrofolate reductase (DHFR). A plasmid-encoded DHFR known as R67 DHFR provides resistance to trimethoprim in bacteria. To better understand the mechanism of this homotetrameric enzyme, a tandem dimer construct was created that linked two monomeric R67 DHFR subunits together and mutated the sequence of residues 66-69 of the first subunit from VQIY to INSF. Using a modified crystn. protocol for this enzyme that included in situ proteolysis using chymotrypsin, the tandem dimer was crystd. and the structure was solved at 1.4 Å resoln. Surprisingly, only wild-type protomers were incorporated into the crystal. Further expts. demonstrated that the variant protomer was selectively degraded by chymotrypsin, although no canonical chymotrypsin cleavage site had been introduced by these mutations.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVCnsrzK&md5=9217e44b24cc69eb9da658b3984a3fad
20. 20
  Ebert, M. C., Morley, K. L., Volpato, J. P., Schmitzer, A. R., and Pelletier, J. N. (2015) Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions. Protein Sci. 24, 495– 507, DOI: 10.1002/pro.2602
  20
  Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions
  Ebert, Maximilian C. C. J. C.; Morley, Krista L.; Volpato, Jordan P.; Schmitzer, Andreea R.; Pelletier, Joelle N.
  Protein Science (2015), 24 (4), 495-507CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
  Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic, trimethoprim. R67 DHFR is genetically and structurally unrelated to Escherichia coli chromosomal DHFR and has an unusual architecture, in that 4 identical protomers form a single sym. active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active site residue has as many as 4 distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although the authors previously demonstrated that no single residue of the native active site was indispensable for function, library selection here revealed a strong bias toward maintenance of 2 native protomers per mutated tetramer. A variety of such "half-native" tetramers were shown to procure native-like catalytic activity, with similar Km values, but kcat values 5- to 33-fold lower, illustrating a high tolerance for active site substitutions. The selected variants showed a reduced thermostability (Tm ∼12° lower), which appeared to result from looser assocn. of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no active site substitutions. These results suggest that the presence of 2 native protomers in the R67 DHFR tetramer is sufficient to provide a native-like catalytic rate and thus ensure cellular proliferation.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkvVOqt7w%253D&md5=7624c11254c18d3f729bf2ea719b5e6c
21. 21
  Baskakov, I. and Bolen, D. W. (1998) Forcing thermodynamically unfolded proteins to fold. J. Biol. Chem. 273, 4831– 4834, DOI: 10.1074/jbc.273.9.4831
  21
  Forcing thermodynamically unfolded proteins to fold
  Baskakov, Ilia; Bolen, D. Wayne
  Journal of Biological Chemistry (1998), 273 (9), 4831-4834CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  A growing no. of biol. important proteins have been identified as fully unfolded or partially disordered. Thus, an intriguing question is whether such proteins can be forced to fold by adding solutes found in the cells of some organisms. Nature has not ignored the powerful effect that the soln. can have on protein stability and has developed the strategy of using specific solutes (called org. osmolytes) to maintain the structure and function of cellular proteins in organisms exposed to denaturing environmental stresses. Here, the authors illustrate the extraordinary capability of one such osmolyte, trimethylamine N-oxide (TMAO), to force two thermodynamically unfolded proteins to fold to native-like species having significant functional activity. In one of these examples, TMAO is shown to increase the population of the native state relative to the denatured ensemble by nearly five orders of magnitude. The ability of TMAO to force thermodynamically unstable proteins to fold presents an opportunity for structure detn. and functional studies of an important emerging class of proteins that have little or no structure without the presence of TMAO.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhs1CgtLo%253D&md5=40a68c935b335c4f86c2de8d1ffd2d88
22. 22
  Parsegian, V. A., Rand, R. P., and Rau, D. C. (2000) Osmotic stress, crowding, preferential hydration, and binding: A comparison of perspectives. Proc. Natl. Acad. Sci. U. S. A. 97, 3987– 3992, DOI: 10.1073/pnas.97.8.3987
  22
  Osmotic stress, crowding, preferential hydration, and binding: a comparison of perspectives
  Parsegian, V. A.; Rand, R. P.; Rau, D. C.
  Proceedings of the National Academy of Sciences of the United States of America (2000), 97 (8), 3987-3992CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  There has been much confusion recently about the relative merits of different approaches, osmotic stress, preferential interaction, and crowding, to describe the indirect effect of solutes on macromol. conformations and reactions. To strengthen all interpretations of measurements and to forestall further unnecessary conceptual or linguistic confusion, we show here how the different perspectives all can be reconciled. Our approach is through the Gibbs-Duhem relation, the universal constraint on the no. of ways it is possible to change the temp., pressure, and chem. potentials of the several components in any thermodynamically defined system. From this general Gibbs-Duhem equation, it is possible to see the equivalence of the different perspectives and even to show the precise identity of the more specialized equations that the different approaches use.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXislSgt70%253D&md5=158b27c5a0e496fe295b4a02385cdd34
23. 23
  Biedermannova, L. and Schneider, B. (2016) Hydration of proteins and nucleic acids: Advances in experiment and theory. A review. Biochim. Biophys. Acta, Gen. Subj. 1860, 1821– 1835, DOI: 10.1016/j.bbagen.2016.05.036
  23
  Hydration of proteins and nucleic acids: Advances in experiment and theory. A review
  Biedermannova, Lada; Schneider, Bohdan
  Biochimica et Biophysica Acta, General Subjects (2016), 1860 (9), 1821-1835CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
  A review. Most biol. processes involve water, and the interactions of biomols. with water affect their structure, function and dynamics. This review summarizes the current knowledge of protein and nucleic acid interactions with water, with a special focus on the biomol. hydration layer. Recent developments in both exptl. and computational methods that can be applied to the study of hydration structure and dynamics are reviewed, including software tools for the prediction and characterization of hydration layer properties. In the last decade, important advances have been made in our understanding of the factors that det. how biomols. and their aq. environment influence each other. Both exptl. and computational methods contributed to the gradually emerging consensus picture of biomol. hydration. An improved knowledge of the structural and thermodn. properties of the hydration layer will enable a detailed understanding of the various biol. processes in which it is involved, with implications for a wide range of applications, including protein-structure prediction and structure-based drug design.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XovVagurs%253D&md5=b87cb044d617b6868eec0e738f272bfc
24. 24
  Rani, P. and Biswas, P. (2015) Diffusion of Hydration Water around Intrinsically Disordered Proteins. J. Phys. Chem. B 119, 13262– 13270, DOI: 10.1021/acs.jpcb.5b07248
  24
  Diffusion of Hydration Water around Intrinsically Disordered Proteins
  Rani, Pooja; Biswas, Parbati
  Journal of Physical Chemistry B (2015), 119 (42), 13262-13270CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
  Hydration water dynamics around globular proteins have attracted considerable attention in the past decades. This work investigates the hydration water dynamics around partially/fully intrinsically disordered proteins (IDPs) and compares it to that of the globular proteins via mol. dynamics (MD) simulations. The translational diffusion of the hydration water is examd. by evaluating the mean-square displacement and the velocity autocorrelation function, while the rotational diffusion is probed through the dipole-dipole time correlation function. The results reveal that the translational and rotational motions of water mols. at the surface of intrinsically disordered proteins/regions are less restricted in comparison to compared to those around globular proteins/ordered regions, which is reflected in their higher diffusion coeff. and lower orientational relaxation time. The restricted mobility of hydration water in the vicinity of the protein leads to a sublinear diffusion in a heterogeneous interface. A pos. correlation between the mean no. of hydrogen bonds and the diffusion coeff. of hydration water implies higher mobility of water mols. at the surface of disordered proteins, which is due to their higher no. of hydrogen bonds. Enhanced hydration water mobility around disordered proteins/regions is also related to their higher hydration capacity, low hydrophobicity, and increased internal protein motions. Thus, we generalize that the intrinsically disordered proteins/regions are assocd. with higher hydration water mobility as compared to globular protein/ordered regions, which may help to elucidate their varied functional specificity.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFyrurnO&md5=51309a05e54693cd4ec53ee220701aca
25. 25
  Rani, P. and Biswas, P. (2015) Local Structure and Dynamics of Hydration Water in Intrinsically Disordered Proteins. J. Phys. Chem. B 119, 10858– 10867, DOI: 10.1021/jp511961c
  25
  Local Structure and Dynamics of Hydration Water in Intrinsically Disordered Proteins
  Rani, Pooja; Biswas, Parbati
  Journal of Physical Chemistry B (2015), 119 (34), 10858-10867CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
  Hydration water around protein surface plays a key role in structure, folding and dynamics of proteins. Intrinsically disordered proteins lack secondary and/or tertiary structure in their native state. Thus, characterizing the local structure and dynamics of hydration water around disordered proteins is challenging for both experimentalists and theoreticians. The local structure, orientation and dynamics of hydration water in the vicinity of intrinsically disordered proteins is investigated through mol. dynamics simulations. The anal. of the hydration capacity reveals that the disordered proteins have much larger binding capacity for hydration water than globular proteins. The surface and radial distribution of water mols. around the disordered proteins depict a similar trend. The local structure of the hydration water evaluated in terms of the tetrahedral order parameter, shows a higher order among the water mols. surrounding disordered proteins/regions. The residence time of water mols. clearly exhibits slow dynamics of hydration water around the surface of disordered proteins/regions as compared to globular proteins. The orientation of water mols. is found to be distinctly different for ordered and disordered proteins/regions. This anal. provides a better insight into the structure and dynamics of hydration water around disordered proteins.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmsVemtLc%253D&md5=7ebac22aa3b9ea87025dfab7fe0be5f6
26. 26
  Park, H. (1997) Creation and characterization of asymmetric mutations in R67 dihydrofolate reductases. Ph.D. Dissertation, University of Tennessee, Knoxville, TN.
  There is no corresponding record for this reference.
27. 27
  Stanley, C., Krueger, S., Parsegian, V. A., and Rau, D. C. (2008) Protein structure and hydration probed by SANS and osmotic stress. Biophys. J. 94, 2777– 2789, DOI: 10.1529/biophysj.107.122697
  27
  Protein structure and hydration probed by SANS and osmotic stress
  Stanley, Christopher; Krueger, Susan; Parsegian, V. Adrian; Rau, Donald C.
  Biophysical Journal (2008), 94 (7), 2777-2789CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
  Interactions governing protein folding, stability, recognition, and activity are mediated by hydration. Here, we use small-angle neutron scattering (SANS) coupled with osmotic stress to investigate the hydration of two proteins, lysozyme and guanylate kinase (GK), in the presence of solutes. By taking advantage of the neutron contrast variation that occurs upon addn. of these solutes, the no. of protein-assocd. (solute-excluded) water mols. can be estd. from changes in both the zero-angle scattering intensity and the radius of gyration. Poly(ethylene glycol) exclusion varies with mol. wt. This sensitivity can be exploited to probe structural features such as the large internal GK cavity. For GK, small-angle neutron scattering is complemented by isothermal titrn. calorimetry with osmotic stress to also measure hydration changes accompanying ligand binding. These results provide a framework for studying other biomol. systems and assemblies using neutron scattering together with osmotic stress.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXjslCjsrk%253D&md5=b3ec7dd2048fd5f6e8e58a3820b81be1
28. 28
  Arnold, O., Bilheux, J. C., Borreguero, J. M., Buts, A., Campbell, S. I., Chapon, L., Doucet, M., Draper, N., Ferraz Leal, R., Gigg, M. A., Lynch, V. E., Markvardsen, A., Mikkelson, D. J., Mikkelson, R. L., Miller, R., Palmen, K., Parker, P., Passos, G., Perring, T. G., Peterson, P. F., Ren, S., Reuter, M. A., Savici, A. T., Taylor, J. W., Taylor, R. J., Tolchenov, R., Zhou, W., and Zikovsky, J. (2014) Mantid-Data analysis and visualization package for neutron scattering and mu SR experiments. Nucl. Instrum. Methods Phys. Res., Sect. A 764, 156– 166, DOI: 10.1016/j.nima.2014.07.029
  28
  Mantid-Data analysis and visualization package for neutron scattering and μ SR experiments
  Arnold, O.; Bilheux, J. C.; Borreguero, J. M.; Buts, A.; Campbell, S. I.; Chapon, L.; Doucet, M.; Draper, N.; Ferraz Leal, R.; Gigg, M. A.; Lynch, V. E.; Markvardsen, A.; Mikkelson, D. J.; Mikkelson, R. L.; Miller, R.; Palmen, K.; Parker, P.; Passos, G.; Perring, T. G.; Peterson, P. F.; Ren, S.; Reuter, M. A.; Savici, A. T.; Taylor, J. W.; Taylor, R. J.; Tolchenov, R.; Zhou, W.; Zikovsky, J.
  Nuclear Instruments & Methods in Physics Research, Section A: Accelerators, Spectrometers, Detectors, and Associated Equipment (2014), 764 (), 156-166CODEN: NIMAER; ISSN:0168-9002. (Elsevier B.V.)
  The Mantid framework is a software soln. developed for the anal. and visualization of neutron scattering and muon spin measurements. The framework is jointly developed by software engineers and scientists at the ISIS Neutron and Muon Facility and the Oak Ridge National Lab. The objectives, functionality and novel design aspects of Mantid are described.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlGntrfL&md5=9a34bb9e366fca0e39091a5c4016ccd7
29. 29
  Svergun, D. I. (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria.. J. Appl. Crystallogr. 25, 495– 503, DOI: 10.1107/S0021889892001663
  29
  Determination of the regularization parameter in indirect-transform methods using perceptual criteria
  Svergun, D. I.
  Journal of Applied Crystallography (1992), 25 (4), 495-503CODEN: JACGAR; ISSN:1600-5767. (International Union of Crystallography)
  A method is proposed for the detn. of the optimum value of the regularization parameter (Lagrange multiplier) when applying indirect transform techniques in small-angle scattering data anal. The method is based on perceptual criteria of what is the best soln. A set of simple criteria is used to construct a total est. describing the quality of the soln. Maximization of the total est. is straightforward. Model computations show the effectiveness of the technique. The method is implemented in the program GNOM.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslaks7vF&md5=98cf53dc2febca54bbbd505f698e1269
30. 30
  Whitten, A. E., Cai, S., and Trewhella, J. (2008) MULCh: modules for the analysis of small-angle neutron contrast variation data from biomolecular assemblies. J. Appl. Crystallogr. 41, 222– 226, DOI: 10.1107/S0021889807055136
  30
  MULCh: modules for the analysis of small-angle neutron contrast variation data from biomolecular assemblies
  Whitten, Andrew E.; Cai, Shuzhi; Trewhella, Jill
  Journal of Applied Crystallography (2008), 41 (1), 222-226CODEN: JACGAR; ISSN:0021-8898. (International Union of Crystallography)
  Small-angle neutron scattering with contrast variation can fill important gaps in our understanding of biomol. assemblies, providing constraints that can aid in the construction of mol. models and in subsequent model refinements. This paper describes the implementation of simple tools for analyzing neutron contrast variation data, accessible via a user-friendly web-based interface (http://www.mmb.usyd.edu.au/NCVWeb/). There are three modules accessible from the website to analyze neutron contrast variation data from bimol. complexes. The first module, Contrast, computes neutron contrasts of each component of the complex required by the other two modules; the second module, Rg, analyses the contrast dependence of the radii of gyration to yield information relating to the size and disposition of each component in the complex; and the third, Compost, decomps. the contrast variation series into composite scattering functions, which contain information regarding the shape of each component of the complex, and their orientation with respect to each other.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmvFymtw%253D%253D&md5=362603873968ba4922479a40906d85c7
31. 31
  Curtis, J. E., Raghunandan, S., Nanda, H., and Krueger, S. (2012) SASSIE: A program to study intrinsically disordered biological molecules and macromolecular ensembles using experimental scattering restraints,. Comput. Phys. Commun. 183, 382– 389, DOI: 10.1016/j.cpc.2011.09.010
  31
  SASSIE: A program to study intrinsically disordered biological molecules and macromolecular ensembles using experimental scattering restraints
  Curtis, Joseph E.; Raghunandan, Sindhu; Nanda, Hirsh; Krueger, Susan
  Computer Physics Communications (2012), 183 (2), 382-389CODEN: CPHCBZ; ISSN:0010-4655. (Elsevier B.V.)
  A program to construct ensembles of biomol. structures that are consistent with exptl. scattering data are described. Specifically, we generate an ensemble of biomol. structures by varying sets of backbone dihedral angles that are then filtered using exptl. detd. restraints to rapidly det. structures that have scattering profiles that are consistent with scattering data. We discuss an application of these tools to predict a set of structures for the HIV-1 Gag protein, an intrinsically disordered protein, that are consistent with small-angle neutron scattering exptl. data. We have assembled these algorithms into a program called SASSIE for structure generation, visualization, and anal. of intrinsically disordered proteins and other macromol. ensembles using neutron and X-ray scattering restraints. Open source software to generate structures of disordered biol. mols. that subsequently allow for the comparison of computational and exptl. results is limiting the use of scattering resources. Starting with an all atom model of a protein, for example, users can input regions to vary dihedral angles, ensembles of structures can be generated. Addnl., simple two-body rigid-body rotations are supported with and without disordered regions. Generated structures can then be used to calc. small-angle scattering profiles which can then be filtered against exptl. detd. data. Filtered structures can be visualized individually or as an ensemble using d. plots. In the modular and expandable program framework the user can easily access our subroutines and structural coordinates can be easily obtained for study using other computational physics methods. The distribution file for this program is over 159 Mbytes and therefore is not delivered directly when download or Email is requested. Instead an html file giving details of how the program can be obtained is sent. Running time varies depending on application. Typically 10 min to 24 h depending on the no. of generated structures.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsV2is7vJ&md5=ad02c0b0220a6da5de20deaab3b1bee7
32. 32
  Webb, B. and Sali, A. (2014) Comparative Protein Structure Modeling Using MODELLER. Current Protocols in Bioinformatics 47, 5.6.1– 5.6.32, DOI: 10.1002/0471250953.bi0506s47
  There is no corresponding record for this reference.
33. 33
  Berendsen, H. J. C., Grigera, J. R., and Straatsma, T. P. (1987) The missing term in effective pair potentials. J. Phys. Chem. 91, 6269– 6271, DOI: 10.1021/j100308a038
  33
  The missing term in effective pair potentials
  Berendsen, H. J. C.; Grigera, J. R.; Straatsma, T. P.
  Journal of Physical Chemistry (1987), 91 (24), 6269-71CODEN: JPCHAX; ISSN:0022-3654.
  Effective pair potentials used for simulations of polar liqs. include the av. effects of polarization. Such potentials are generally adjusted to produce the exptl. heat of vaporization. It has not been recognized before that the self-energy term inherent in any polarizable model should be included in effective pair potentials as well. Inclusion of the self-energy correction with a consequent reparametrization of the simple point charge model of water yields an improvement of the effective pair potential for water, as exemplified by d., radial distribution functions, and diffusion const.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXmt1els7w%253D&md5=6668667f6252092fc001ae8d422ebb94
34. 34
  Ramanathan, A., Savol, A. J., Langmead, C. J., Agarwal, P. K., and Chennubhotla, C. S. (2011) Discovering conformational sub-states relevant to protein function. PLoS One 6, e15827 DOI: 10.1371/journal.pone.0015827
  34
  Discovering conformational sub-states relevant to protein function
  Ramanathan, Arvind; Savol, Andrej J.; Langmead, Christopher J.; Agarwal, Pratul K.; Chennubhotla, Chakra S.
  PLoS One (2011), 6 (1), e15827CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
  Background: Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant. However, low populations in these sub-states and the transient nature of conformational transitions between these sub-states present significant challenges for their identification and characterization. Methods and Findings: To overcome these challenges we have developed a new computational technique, quasi-anharmonic anal. (QAA). QAA utilizes higher-order statistics of protein motions to identify sub-states in the conformational landscape. Further, the focus on anharmonicity allows identification of conformational fluctuations that enable transitions between sub-states. QAA applied to equil. simulations of human ubiquitin and T4 lysozyme reveals functionally relevant sub-states and protein motions involved in mol. recognition. In combination with a reaction pathway sampling method, QAA characterizes conformational sub-states assocd. with cis/trans peptidyl-prolyl isomerization catalyzed by the enzyme cyclophilin A. In these 3 proteins, QAA allows identification of conformational sub-states, with crit. structural and dynamical features relevant to protein function. Conclusions: Overall, QAA provides a novel framework to intuitively understand the biophys. basis of conformational diversity and its relevance to protein function.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhvVKksro%253D&md5=7ecb8360e065745b1cdae3f4b23968d8
35. 35
  Case, D. A., Babin, V., Berryman, J. T., Betz, R. M., Cai, Q., Cerutti, D. S., Cheatham, T. E., III, Darden, T. A., Duke, R. E., Gohlke, H., Goetz, A. W., Gusarov, S., Homeyer, N., Janowski, P., Kaus, J., Kolossváry, I., Kovalenko, A., Lee, T. S., LeGrand, S., Luchko, T., Luo, R., Madej, B., Merz, K. M., Paesani, F., Roe, D. R., Roitberg, A., Sagui, C., Salomon-Ferrer, R., Seabra, G., Simmerling, C. L., Smith, W., Walker, R. C., Wang, J., Wolf, R. M., Wu, X., and Kollman, P. A. (2014) AMBER14, University of California, San Francisco.
  There is no corresponding record for this reference.
36. 36
  Bradrick, T. D., Beechem, J. M., and Howell, E. E. (1996) Unusual binding stoichiometries and cooperativity are observed during binary and ternary complex formation in the single active pore of R67 dihydrofolate reductase, a D₂ symmetric protein. Biochemistry 35, 11414– 11424, DOI: 10.1021/bi960205d
  36
  Unusual Binding Stoichiometries and Cooperativity Are Observed during Binary and Ternary Complex Formation in the Single Active Pore of R67 Dihydrofolate Reductase, a D2 Symmetric Protein
  Bradrick, Thomas D.; Beechem, Joseph M.; Howell, Elizabeth E.
  Biochemistry (1996), 35 (35), 11414-11424CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  R67 dihydrofolate reductase (DHFR) is an R-plasmid-encoded enzyme that confers resistance to the antibacterial drug, trimethoprim. This DHFR variant is not homologous in either sequence or structure to chromosomal DHFRs. A recent crystal structure of the active tetrameric species describes a single active site pore that traverses the length of the protein (Narayana et al., 1995). Related sites (due to a 222 symmetry element at the center of the active site pore) are used for binding of ligands, i.e., each half-pore can accommodate either the substrate, dihydrofolate, or the cofactor, NADPH, although dihydrofolate and NADPH are bound differently. Ligand binding in R67 DHFR was evaluated using time-resolved fluorescence anisotropy and isothermal titrn. calorimetry techniques. Under binary complex conditions, two mols. of either NADPH, folate, dihydrofolate, or N10 propargyl-5,8-dideazafolate (CB3717) can be bound. Binding of NADPH displays neg. cooperativity, binding of either folate or dihydrofolate shows pos. cooperativity, and binding of CB3717 shows two identical sites. Any asymmetry introduced by binding of one ligand is proposed to induce the cooperativity assocd. with binding of the second ligand. Evaluation of ternary complex formation demonstrates that one mol. of folate binds to a 1:1 mixt. of R67 DHFR + NADPH. These binding results indicate a max. of two ligands bind in the pore. A mechanism describing catalysis is proposed that is consistent with the binding results.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XkvVGqsbc%253D&md5=f85af8ec7de91eb41c60c55aae8604a9
37. 37
  Lin, L. N., Brandts, J. F., Brandts, J. M., and Plotnikov, V. (2002) Determination of the volumetric properties of proteins and other solutes using pressure perturbation calorimetry. Anal. Biochem. 302, 144– 160, DOI: 10.1006/abio.2001.5524
  37
  Determination of the volumetric properties of proteins and other solutes using pressure perturbation calorimetry
  Lin, Lung-Nan; Brandts, John F.; Brandts, J. Michael; Plotnikov, Valerian
  Analytical Biochemistry (2002), 302 (1), 144-160CODEN: ANBCA2; ISSN:0003-2697. (Academic Press)
  Pressure perturbation calorimetry is a new technique that measures the heat change in a soln. that results when the pressure above the soln. is changed. When used in a differential calorimeter contg. a dil. soln. of solute in the sample cell and the corresponding buffer in the ref. cell, the measured differential heat can be used to calc. the thermal coeff. of expansion of the partial vol. of the solute,. For proteins in dil. aq. soln., is dominated by a temp.-dependent contribution arising from the interaction of protein groups with water at the protein-solvent interface. This arises due to the effect of the protein groups on the hydrogen-bonded structure of water, and thereby clearly differentiates between structure-making hydrophobic groups and structure-breaking hydrophilic groups. This solvation contribution to can be accentuated in solvents having more structure (deuterium oxide) than water and attenuated in solvents having less structure (2.8 M guanidinium sulfate). Six different proteins (chymotrypsinogen, pepsinogen, lysozyme, bovine pancreatic trypsin inhibitor, RNase A, and T4 lysozyme) were examd. carefully by this technique, allowing ests. of various volumetric parameters including the vol. change resulting from thermal unfolding of each protein. For RNase A, results obtained in both water and deuterium oxide led to an est. of the accessible surface area of the native protein of ∼45% relative to the fully reduced unfolded protein. Also, it was also found that ligand binding to RNase A led to changes in, suggesting a burial of some surface area in the ligand-protein complex. (c) 2002 Academic Press.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhtFels78%253D&md5=15248e92350b9659d4c7934605921e2f
38. 38
  Mitra, L., Smolin, N., Ravindra, R., Royer, C., and Winter, R. (2006) Pressure perturbation calorimetric studies of the solvation properties and the thermal unfolding of proteins in solution–experiments and theoretical interpretation. Phys. Chem. Chem. Phys. 8, 1249– 1265, DOI: 10.1039/b516608j
  There is no corresponding record for this reference.
39. 39
  Keller, S., Vargas, C., Zhao, H., Piszczek, G., Brautigam, C. A., and Schuck, P. (2012) High-precision isothermal titration calorimetry with automated peak-shape analysis. Anal. Chem. 84, 5066– 5073, DOI: 10.1021/ac3007522
  39
  High-Precision Isothermal Titration Calorimetry with Automated Peak-Shape Analysis
  Keller, Sandro; Vargas, Carolyn; Zhao, Huaying; Piszczek, Grzegorz; Brautigam, Chad A.; Schuck, Peter
  Analytical Chemistry (Washington, DC, United States) (2012), 84 (11), 5066-5073CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
  Isothermal titrn. calorimetry (ITC) is a powerful classical method that enables researchers in many fields to study the thermodn. of mol. interactions. Primary ITC data comprise the temporal evolution of differential power reporting the heat of reaction during a series of injections of aliquots of a reactant into a sample cell. By integration of each injection peak, an isotherm can be constructed of total changes in enthalpy as a function of changes in soln. compn., which is rich in thermodn. information on the reaction. However, the signals from the injection peaks are superimposed by the stochastically varying time-course of the instrumental baseline power, limiting the precision of ITC isotherms. The authors describe a method for automated peak assignment based on peak-shape anal. via singular value decompn. in combination with detailed least-squares modeling of local pre- and post- injection baselines. This approach can effectively filter out contributions of short-term noise and adventitious events in the power trace. This method also provides, for the first time, statistical error ests. for the individual isotherm data points. In turn, this results in improved detection limits for high-affinity or low-enthalpy binding reactions and significantly higher precision of the derived thermodn. parameters.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmtVGlsbs%253D&md5=bb157e956fa9cef94a88974d733cad1b
40. 40
  Timson, M. J., Duff, M. R., Jr., Dickey, G., Saxton, A. M., Reyes-De-Corcuera, J. I., and Howell, E. E. (2013) Further studies on the role of water in R67 dihydrofolate reductase. Biochemistry 52, 2118– 2127, DOI: 10.1021/bi301544k
  40
  Further Studies on the Role of Water in R67 Dihydrofolate Reductase
  Timson, Mary Jane; Duff, Michael R.; Dickey, Greyson; Saxton, Arnold M.; Reyes-De-Corcuera, Jose I.; Howell, Elizabeth E.
  Biochemistry (2013), 52 (12), 2118-2127CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  Previous osmotic pressure studies of two nonhomologous dihydrofolate reductase (DHFR) enzymes found tighter binding of the NADP cofactor upon addn. of neutral osmolytes. This result is consistent with water release accompanying binding. In contrast, osmotic stress studies found weaker binding of the dihydrofolate (DHF) substrate for both type I and type II DHFRs in the presence of osmolytes; this observation can be explained if dihydrofolate interacts with osmolytes and shifts the equil. from the enzyme-bound state toward the unbound substrate. NMR expts. support this hypothesis, finding that osmolytes interact with dihydrofolate. To consider binding without added osmolytes, a high-pressure approach was used. In this study, the type II enzyme, R67 DHFR, was subjected to high hydrostatic pressure (HHP). Both enzyme activity and fluorescence measurements find the protein tolerates pressures up to 200 MPa. Binding of the cofactor to R67 DHFR weakens with increasing pressure, and a pos. assocn. vol. of 11.4 ± 0.5 cm3/mol was measured. Addnl., an activation vol. of 3.3 ± 0.5 cm3/mol describing kcat/Km(DHF) was detd. from progress curve anal. Results from these HHP expts. suggest water release accompanies binding of both the cofactor and DHF to R67 DHFR. In an addnl. set of expts., isothermal titrn. calorimetry studies in H2O and D2O find that water reorganization dominates the enthalpy assocd. with binding of DHF to R67 DHFR·NADP+, while no obvious effects occur for cofactor binding. The combined results indicate that water plays an active role in ligand binding to R67 DHFR.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjtlGht7c%253D&md5=222ea1b1b4967fa95385d5cf63fa4ade
41. 41
  Receveur-Brechot, V. and Durand, D. (2012) How random are intrinsically disordered proteins? A small angle scattering perspective,. Curr. Protein Pept. Sci. 13, 55– 75, DOI: 10.2174/138920312799277901
  41
  How random are intrinsically disordered proteins? A small angle scattering perspective
  Receveur-Brechot, Veronique; Durand, Dominique
  Current Protein and Peptide Science (2012), 13 (1), 55-75CODEN: CPPSCM; ISSN:1389-2037. (Bentham Science Publishers Ltd.)
  A review. While the crucial role of intrinsically disordered proteins (IDPs) in the cell cycle is now recognized, deciphering their mol. mode of action at the structural level still remains highly challenging and requires a combination of many biophys. approaches. Among them, small angle X-ray scattering (SAXS) has been extremely successful in the last decade and has become an indispensable technique for addressing many of the fundamental questions regarding the activities of IDPs. After introducing some exptl. issues specific to IDPs and in relation to the latest tech. developments, this article presents the interest of the theory of polymer physics to evaluate the flexibility of fully disordered proteins. The different strategies to obtain 3-dimensional models of IDPs, free in soln. and assocd. in a complex, are then reviewed. Indeed, recent computational advances have made it possible to readily ext. max. information from the scattering curve with a special emphasis on highly flexible systems, such as multidomain proteins and IDPs. Furthermore, integrated computational approaches now enable the generation of ensembles of conformers to translate the unique flexible characteristics of IDPs by taking into consideration the constraints of more and more various complementary expt. In particular, a combination of SAXS with high-resoln. techniques, such as x-ray crystallog. and NMR, allows us to provide reliable models and to gain unique structural insights about the protein over multiple structural scales. The latest neutron scattering expts. also promise new advances in the study of the conformational changes of macromols. involving more complex systems.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkvFamtLw%253D&md5=9bb9c34254af874c361317049c577d3c
42. 42
  Svergun, D. I., Richard, S., Koch, M. H., Sayers, Z., Kuprin, S., and Zaccai, G. (1998) Protein hydration in solution: experimental observation by x-ray and neutron scattering. Proc. Natl. Acad. Sci. U. S. A. 95, 2267– 2272, DOI: 10.1073/pnas.95.5.2267
  42
  Protein hydration in solution: experimental observation by x-ray and neutron scattering
  Svergun, D. I.; Richard, S.; Koch, M. H. J.; Sayers, Z.; Kuprin, S.; Zaccai, G.
  Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (5), 2267-2272CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  The structure of the protein-solvent interface is the subject of controversy in theor. studies and requires direct exptl. characterization. Three proteins with known at. resoln. crystal structure (lysozyme, Escherichia coli thioredoxin reductase, and protein R1 of E. coli ribonucleotide reductase) were investigated in parallel by x-ray and neutron scattering in H2O and D2O solns. The anal. of the protein-solvent interface is based on the significantly different contrasts for the protein and for the hydration shell. The results point to the existence of a first hydration shell with an av. d. ≈10% larger than that of the bulk solvent in the conditions studied. Comparisons with the results of other studies suggest that this may be a general property of aq. interfaces.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhsleitrw%253D&md5=415ed528ad555452e1b8fb119353c1af
43. 43
  Rambo, R. P. and Tainer, J. A. (2013) Accurate assessment of mass, models and resolution by small-angle scattering. Nature 496, 477– 481, DOI: 10.1038/nature12070
  43
  Accurate assessment of mass, models and resolution by small-angle scattering
  Rambo, Robert P.; Tainer, John A.
  Nature (London, United Kingdom) (2013), 496 (7446), 477-481CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
  Modern small-angle scattering (SAS) expts. with X-rays or neutrons provide a comprehensive, resoln.-limited observation of the thermodn. state. However, methods for evaluating mass and validating SAS-based models and resoln. have been inadequate. Here we define the vol. of correlation, Vc, a SAS invariant derived from the scattered intensities that is specific to the structural state of the particle, but independent of concn. and the requirements of a compact, folded particle. We show that Vc defines a ratio, QR, that dets. the mol. mass of proteins or RNA ranging from 10 to 1,000 kilodaltons. Furthermore, we propose a statistically robust method for assessing model-data agreements (χ2free) akin to cross-validation. Our approach prevents over-fitting of the SAS data and can be used with a newly defined metric, RSAS, for quant. evaluation of resoln. Together, these metrics (Vc, QR, χ2free and RSAS) provide anal. tools for unbiased and accurate macromol. structural characterizations in soln.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXms1Wgu7o%253D&md5=ca998ec661eaa9015e93c4f52c083bce
44. 44
  Santner, A. A., Croy, C. H., Vasanwala, F. H., Uversky, V. N., Van, Y. Y., and Dunker, A. K. (2012) Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression. Biochemistry 51, 7250– 7262, DOI: 10.1021/bi300653m
  44
  Sweeping Away Protein Aggregation with Entropic Bristles: Intrinsically Disordered Protein Fusions Enhance Soluble Expression
  Santner, Aaron A.; Croy, Carrie H.; Vasanwala, Farha H.; Uversky, Vladimir N.; Van, Ya-Yue J.; Dunker, A. Keith
  Biochemistry (2012), 51 (37), 7250-7262CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both a large favorable surface area for water interactions and large excluded vols. around the partner. By extending away from the partner and sweeping out large mols., EBs can allow the target protein to fold free from interference. Using both naturally occurring and artificial polypeptides, the authors demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have a low level of sequence complexity and a high net charge but are diversified by distinctive amino acid compns. and lengths. Using 6xHis fusions as controls, sol. protein expression enhancements from 65% (EB60A) to 100% (EB250) were obsd. for a 20-protein portfolio. Addnl., these EBs were able to more effectively solubilize targets compared to frequently used fusions such as maltose-binding protein, glutathione S-transferase, thioredoxin, and N use substance A. Finally, although these EBs possess very distinct physiochem. properties, they did not perturb the structure, conformational stability, or function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically disordered fusions and presents a promising technol. and complementary resource for researchers attempting to solubilize recalcitrant proteins.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1Kitr3F&md5=abf5e6e0125744b920386c7922a0588b
45. 45
  Fried, M. G., Stickle, D. F., Smirnakis, K. V., Adams, C., MacDonald, D., and Lu, P. (2002) Role of hydration in the binding of lac repressor to DNA. J. Biol. Chem. 277, 50676– 50682, DOI: 10.1074/jbc.M208540200
  45
  Role of Hydration in the Binding of lac Repressor to DNA
  Fried, Michael G.; Stickle, Douglas F.; Smirnakis, Karen Vossen; Adams, Claire; MacDonald, Douglas; Lu, Ponzy
  Journal of Biological Chemistry (2002), 277 (52), 50676-50682CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  The osmotic stress technique was used to measure changes in macromol. hydration that accompany binding of wild-type Escherichia coli lactose (lac) repressor to its regulatory site (operator O1) in the lac promoter and its transfer from site O1 to nonspecific DNA. Binding at O1 is accompanied by the net release of 260±32 water mols. If all are released from macromol. surfaces, this result is consistent with a net redn. of solvent-accessible surface area of 2370±550 Å. This area is only slightly smaller than the macromol. interface calcd. for a cryst. repressor dimer-O1 complex but is significantly smaller than that for the corresponding complex with the sym. optimized Osym operator. The transfer of repressor from site O1 to nonspecific DNA is accompanied by the net uptake of 93±10 water mols. Together these results imply that formation of a nonspecific complex is accompanied by the net release of 165±43 water mols. The enhanced stabilities of repressor-DNA complexes with increasing osmolality may contribute to the ability of Escherichia coli cells to tolerate dehydration and/or high external salt concns.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xps12nt7o%253D&md5=a3f2035c2b60dd88e07c86ae3a440cb7
46. 46
  Tsodikov, O. V., Record, M. T., Jr., and Sergeev, Y. V. (2002) Novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature. J. Comput. Chem. 23, 600– 609, DOI: 10.1002/jcc.10061
  46
  Novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature
  Tsodikov, Oleg V.; Record, M. Thomas, Jr.; Sergeev, Yuri V.
  Journal of Computational Chemistry (2002), 23 (6), 600-609CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
  New computer programs, SurfRace and FastSurf, perform fast calcns. of the solvent accessible and mol. (solvent excluded) surface areas of macromols. Program SurfRace also calcs. the areas of cavities inaccessible from the outside. We introduce the definition of av. curvature of mol. surface and calc. av. mol. surface curvatures for each atom in a structure. All surface area and curvature calcns. are analytic and therefore yield exact values of these quantities. High calcn. speed of this software is achieved primarily by avoiding computationally expensive math. procedures wherever possible and by efficient handling of surface data structures. The programs are written initially in the language C for PCs running Windows 2000/98/NT, but their code is portable to other platforms with only minor changes in input-output procedures. The algorithm is robust and does not ignore either multiplicity or degeneracy of at. overlaps. Fast, memory-efficient and robust execution make this software attractive for applications both in computationally expensive energy minimization algorithms, such as docking or mol. dynamics simulations, and in stand-alone surface area and curvature calcns.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjtFKrtLY%253D&md5=52b6b7f3c22ecb6d541167ab361501b1
47. 47
  Zhuang, P., Eisenstein, E., and Howell, E. E. (1994) Equilibrium folding studies of tetrameric R67 dihydrofolate reductase. Biochemistry 33, 4237– 4244, DOI: 10.1021/bi00180a018
  47
  Equilibrium folding studies of tetrameric R67 dihydrofolate reductase
  Zhuang, Ping; Eisenstein, Edward; Howell, Elizabeth E.
  Biochemistry (1994), 33 (14), 4237-44CODEN: BICHAW; ISSN:0006-2960.
  R67 dihydrofolate reductase (DHFR) is an R-plasmid encoded enzyme that confers resistance to the antibacterial drug trimethoprim. This enzyme is not homologous in sequence or structure to chromosomal DHFRs. Equil. folding of tetrameric R67 DHFR was studied and found to be fully reversible. Formation of an inactive intermediate was assayed by loss of enzyme activity. Denaturation of the intermediate was monitored by concurrent changes in fluorescence and CD signals. Both transitions are protein concn. dependent. A simple model fitting these data is tetramer ↹ 2 dimers ↹ 4 unfolded monomers. No evidence for folded monomer was found. Global fitting of all the folding data yielded a ΔGH2O of -9.63 kcal/mol for the initial transition and a ΔGH2O of -12.35 kcal/mol for the second transition. In addn., thermal unfolding of tetrameric R67 DHFR was found to be reversible. A folding intermediate also occurred during thermal unfolding as evidenced by the asym. endotherms and a ΔHcalorimetric/ΔHvan't Hoff ratio of 2.1.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXivFarsLw%253D&md5=e3972a1fba7dbd915381fd226449d670
48. 48
  Permyakov, S. (2012) Differential Scanning Microcalorimetry of Intrinsically Disordered Proteins. In Intrinsically Disordered Protein Analysis, Vol. 2, Methods and Experimental Tools (Uversky, V. L., and Dunker, A. K., Eds.) pp 283– 296, Springer, Dordrecht, The Netherlands.
  There is no corresponding record for this reference.
49. 49
  Permyakov, S. E., Bakunts, A. G., Denesyuk, A. I., Knyazeva, E. L., Uversky, V. N., and Permyakov, E. A. (2008) Apo-parvalbumin as an intrinsically disordered protein. Proteins: Struct., Funct., Genet. 72, 822– 836, DOI: 10.1002/prot.21974
  There is no corresponding record for this reference.
50. 50
  Parsegian, V. A., Rand, R. P., and Rau, D. C. (1995) Macromolecules and water: probing with osmotic stress. Methods Enzymol. 259, 43– 94, DOI: 10.1016/0076-6879(95)59039-0
  50
  Macromolecules and water: probing with osmotic stress
  Parsegian, V. Adrian; Rand, R. Peter; Rau, Donald C.
  Methods in Enzymology (1995), 259 (Energetics of Biological Macromolecules), 43-94CODEN: MENZAU; ISSN:0076-6879. (Academic)
  An intuitive picture of osmotic stress on macromols. is given. The authors also illustrate the method as it is applied to 4 kinds of processes: (1)ionic channel opening/closing, (2)enzyme/substrate assocn. and turnover, (3)mol. binding, and (4)long-range interaction.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xht1entbw%253D&md5=1c37a31f8ecfbc0cb05cf27e508d131d
51. 51
  Royer, C. A. (2002) Revisiting volume changes in pressure-induced protein unfolding. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1595, 201– 209, DOI: 10.1016/S0167-4838(01)00344-2
  51
  Revisiting volume changes in pressure-induced protein unfolding
  Royer, Catherine A.
  Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (2002), 1595 (1-2), 201-209CODEN: BBAEDZ; ISSN:0167-4838. (Elsevier B.V.)
  A review. It has long been known that the application of hydrostatic pressure generally leads to the unfolding of proteins. Despite a relatively large no. of reports in the literature over the past few decades, there has been great confusion over the sign and magnitude as well as the fundamental factors contributing to vol. effects in protein conformational transitions. It is the goal of this review to present and discuss the results obtained concerning the sign and magnitude of the vol. changes accompanying the unfolding of proteins. The vast majority of cases point to a significant decrease in vol. upon unfolding. Nonetheless, there is evidence that, due to differences in the thermal expansivity of the folded and unfolded states of proteins reported in a half dozen manuscripts, that the sign of the vol. change may become pos. at higher temps.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xjt1Sku7g%253D&md5=0a0bd5797b5bc69072f5cdb57b0a931a
52. 52
  Zhai, Y., Okoro, L., Cooper, A., and Winter, R. (2011) Applications of pressure perturbation calorimetry in biophysical studies. Biophys. Chem. 156, 13– 23, DOI: 10.1016/j.bpc.2010.12.010
  There is no corresponding record for this reference.
53. 53
  Ravindra, R., Royer, C., and Winter, R. (2004) Pressure perturbation calorimetic studies of the solvation properties and the thermal unfolding of Staphylococcal nuclease. Phys. Chem. Chem. Phys. 6, 1952– 1961, DOI: 10.1039/b314172a
  There is no corresponding record for this reference.
54. 54
  Eisenberg, D., Weiss, R. M., Terwilliger, T. C., and Wilcox, W. (1982) Hydrophobic moments and protein structure. Faraday Symp. Chem. Soc. 17, 109– 120, DOI: 10.1039/fs9821700109
  There is no corresponding record for this reference.
55. 55
  Wang, A. and Bolen, D. W. (1997) A naturally occurring protective system in urea-rich cells: mechanism of osmolyte protection of proteins against urea denaturation,. Biochemistry 36, 9101– 9108, DOI: 10.1021/bi970247h
  There is no corresponding record for this reference.
56. 56
  Bhojane, P. P., Duff, M. R., Jr., Bafna, K., Rimmer, G. P., Agarwal, P. K., and Howell, E. E. (2016) Aspects of Weak Interactions between Folate and Glycine Betaine. Biochemistry 55, 6282– 6294, DOI: 10.1021/acs.biochem.6b00873
  56
  Aspects of Weak Interactions between Folate and Glycine Betaine
  Bhojane, Purva P.; Duff, Michael R.; Bafna, Khushboo; Rimmer, Gabriella P.; Agarwal, Pratul K.; Howell, Elizabeth E.
  Biochemistry (2016), 55 (45), 6282-6294CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  Folic acid, or vitamin B9, is an important compd. in 1C metab. Previous studies have found weaker binding of dihydrofolate to dihydrofolate reductase in the presence of osmolytes. In other words, osmolytes are more difficult to remove from the dihydrofolate solvation shell than water; this shifts the equil. toward the free ligand and protein species. Here, vapor pressure osmometry was used to explore the interaction of folate with the model osmolyte, glycine betaine. This method yielded a preferential interaction potential (μ23/RT value). This value was concn.-dependent as folate dimerizes. The μ23/RT value also tracked the deprotonation of folate's N3-O4 keto-enol group, yielding a pKa of 8.1. To det. which folate atoms interact most strongly with betaine, the interaction of heterocyclic arom. compds. (as well as other small mols.) with betaine was monitored. Using an accessible surface area approach coupled with osmometry measurements, deconvolution of the μ23/RT value into α values for atom types was achieved. This allowed prediction of μ23/RT values for larger mols. such as folate. Mol. dynamics simulations of folate showed a variety of structures from extended to L-shaped. These conformers possessed μ23/RT values from -0.18 to 0.09 m-1, where a neg. value indicates a preference for solvation by betaine and a pos. value indicates a preference for water. This range of values was consistent with values obsd. in osmometry and soly. expts. As the av. predicted folate μ23/RT value was near zero, this indicated that folate interacts almost equally well with betaine and water. Specifically, the glutamate tail prefers to interact with water while the arom. rings prefer betaine. In general, the more protonated species in this small mol. survey interacted better with betaine as they provided a source of H atoms (betaine is not a H-bond donor). Upon deprotonation of the small mol., the preference swings toward water interaction due to its H-bond donating capacities.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslagsbjP&md5=88ddfed96ee7bf5fa074f4309684df52
57. 57
  Capp, M. W., Pegram, L. M., Saecker, R. M., Kratz, M., Riccardi, D., Wendorff, T., Cannon, J. G., and Record, M. T., Jr (2009) Interactions of the osmolyte glycine betaine with molecular surfaces in water: thermodynamics, structural interpretation, and prediction of m-values. Biochemistry 48, 10372– 10379, DOI: 10.1021/bi901273r
  57
  Interactions of the Osmolyte Glycine Betaine with Molecular Surfaces in Water: Thermodynamics, Structural Interpretation, and Prediction of m-Values
  Capp, Michael W.; Pegram, Laurel M.; Saecker, Ruth M.; Kratz, Megan; Riccardi, Demian; Wendorff, Timothy; Cannon, Jonathan G.; Record, M. Thomas
  Biochemistry (2009), 48 (43), 10372-10379CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  Noncovalent self-assembly of biopolymers is driven by mol. interactions between functional groups on complementary biopolymer surfaces, replacing interactions with water. Since individually these interactions are comparable in strength to interactions with water, they have been difficult to quantify. Solutes (osmolytes, denaturants) often exert large effects on these self-assembly interactions, detd. in sign and magnitude by how well the solute competes with water to interact with the relevant biopolymer surfaces. Here, an osmometric method and a water-accessible surface area (ASA) anal. are developed to quantify and interpret the interactions of the remarkable osmolyte glycine betaine (GB) with mol. surfaces in water. We find that GB, lacking hydrogen bond donors, is unable to compete with water to interact with anionic and amide oxygens; this explains its effectiveness as an osmolyte in the Escherichia coli cytoplasm. GB competes effectively with water to interact with amide and cationic nitrogens (hydrogen bonding) and esp. with arom. hydrocarbon (cation-π). The large stabilizing effect of GB on lac repressor-lac operator binding is predicted quant. from ASA information and shown to result largely from dehydration of anionic DNA phosphate oxygens in the protein-DNA interface. The incorporation of these results into theor. and computational analyses will likely improve the ability to accurately model intra- and interprotein interactions. Addnl., these results pave the way for development of solutes as kinetic/mechanistic and thermodn. probes of conformational changes and formation/disruption of mol. interfaces that occur in the steps of biomol. self-assembly processes.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1eiurjE&md5=f2b13610ab3cacbd256dbfcad93b9758
58. 58
  Torres, A. M., Grieve, S. M., Chapman, B. E., and Kuchel, P. W. (1997) Strong and weak binding of water to proteins studied by NMR triple-quantum filtered relaxation spectroscopy of (17)O-water. Biophys. Chem. 67, 187– 198, DOI: 10.1016/S0301-4622(97)00039-2
  There is no corresponding record for this reference.
59. 59
  Yang, P. H. and Rupley, J. A. (1979) Protein–water interactions. Heat capacity of the lysozyme–water system. Biochemistry 18, 2654– 2661, DOI: 10.1021/bi00579a035
  There is no corresponding record for this reference.
60. 60
  Pethig, R. (1992) Protein-water interactions determined by dielectric methods. Annu. Rev. Phys. Chem. 43, 177– 205, DOI: 10.1146/annurev.pc.43.100192.001141
  60
  Protein-water interactions determined by dielectric methods
  Pethig, R.
  Annual Review of Physical Chemistry (1992), 43 (), 177-205CODEN: ARPLAP; ISSN:0066-426X.
  A review with 106 refs. Dielec. theory and measurements, soln. studies, and solid-state studies are discussed.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXjsFahsA%253D%253D&md5=fffdae728e861942e5f4ce8a478c1b5c
61. 61
  Kodandapani, R., Suresh, C. G., and Vijayan, M. (1990) Crystal structure of low humidity tetragonal lysozyme at 2.1-A resolution. Variability in hydration shell and its structural consequences. J. Biol. Chem. 265, 16126– 16131
  There is no corresponding record for this reference.
62. 62
  Shimizu, S. and Matubayasi, N. (2014) Preferential solvation: dividing surface vs excess numbers. J. Phys. Chem. B 118, 3922– 3930, DOI: 10.1021/jp410567c
  62
  Preferential Solvation: Dividing Surface vs Excess Numbers
  Shimizu, Seishi; Matubayasi, Nobuyuki
  Journal of Physical Chemistry B (2014), 118 (14), 3922-3930CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
  A review. How do osmolytes affect the conformation and configuration of supramol. assembly, such as ion channel opening and actin polymn.. The key to the answer lies in the excess solvation nos. of water and osmolyte mols.; these nos. are determinable solely from exptl. data, as guaranteed by the phase rule, as we show through the exact soln. theory of Kirkwood and Buff (KB). The osmotic stress technique (OST), in contrast, purposes to yield alternative hydration nos. through the use of the dividing surface borrowed from the adsorption theory. However, we show (i) OST is equiv., when it becomes exact, to the crowding effect in which the osmolyte exclusion dominates over hydration; (ii) crowding is not the universal driving force of the osmolyte effect (e.g., actin polymn.); (iii) the dividing surface for solvation is useful only for crowding, unlike in the adsorption theory which necessitates its use due to the phase rule. KB thus clarifies the true meaning and limitations of the older perspectives on preferential solvation (such as solvent binding models, crowding, and OST), and enables excess no. detn. without any further assumptions.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXltlymtbk%253D&md5=be216ea6a9b539f63088dd901954b397
63. 63
  Zhou, H. X., Rivas, G. N., and Minton, A. P. (2008) Macromolecular crowding and confinement: Biochemical, biophysical, and potential physiological consequences. Annu. Rev. Biophys. 37, 375– 397, DOI: 10.1146/annurev.biophys.37.032807.125817
  63
  Macromolecular crowding and confinement: Biochemical, biophysical, and potential physiological consequences
  Zhou, Huan-Xiang; Rivas, German; Minton, Allen P.
  Annual Review of Biophysics (2008), 37 (), 375-397CODEN: ARBNCV ISSN:. (Annual Reviews Inc.)
  A review. Expected and obsd. effects of vol. exclusion on the free energy of rigid and flexible macromols. in crowded and confined systems, and consequent effects of crowding and confinement on macromol. reaction rates and equil. are summarized. Findings from relevant theor./simulation and exptl. literature published from 2004 onward are reviewed. Addnl. complexity arising from the heterogeneity of local environments in biol. media, and the presence of nonspecific interactions between macromols. over and above steric repulsion, are discussed. Theor. and exptl. approaches to the characterization of crowding- and confinement-induced effects in systems approaching the complexity of living organisms are suggested.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnsVGlurg%253D&md5=2e1fe7edb342b273b68868b02a8d137b
64. 64
  Courtenay, E. S., Capp, M. W., Anderson, C. F., and Record, M. T., Jr (2000) Vapor pressure osmometry studies of osmolyte-protein interactions: implications for the action of osmoprotectants in vivo and for the interpretation of ″osmotic stress″ experiments in vitro. Biochemistry 39, 4455– 4471, DOI: 10.1021/bi992887l
  64
  Vapor Pressure Osmometry Studies of Osmolyte-Protein Interactions: Implications for the Action of Osmoprotectants in Vivo and for the Interpretation of "Osmotic Stress" Experiments in Vitro
  Courtenay, E. S.; Capp, M. W.; Anderson, C. F.; Record, M. T., Jr.
  Biochemistry (2000), 39 (15), 4455-4471CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  To interpret or predict the in vivo and in vitro responses of biopolymer processes to changes in solute concn. and coupled changes in water activity (osmotic stress), a quant. understanding of the thermodn. consequences of interactions of solutes and water with biopolymer surfaces is required. Toward this end, we report isoosmolal preferential interaction coeffs. (Γμ1) detd. by vapor pressure osmometry (VPO) over a wide range of concns. for interactions of native bovine serum albumin (BSA) with six small solutes. These solutes include Escherichia coli cytoplasmic osmolytes [potassium glutamate (K+Glu-), trehalose], E. coli osmoprotectants (proline, glycine betaine), and also glycerol and trimethylamine N-oxide (TMAO). For all six solutes, Γμ1 and the corresponding dialysis preferential interaction coeff. Γμ1,μ3 (both calcd. from the VPO data) are neg.; Γμ1,μ3 is proportional to bulk solute molality (m3bulk) at least up to 1 m (molal). Neg. values of Γμ1,μ3 indicate preferential exclusion of these solutes from a BSA soln. at dialysis equil. and correspond to local concns. of these solutes in the vicinity of BSA which are lower than their bulk concns. Of the solutes investigated, betaine is the most excluded (Γμ1,μ3/m3bulk = -49 ± 1 m-1); glycerol is the least excluded (Γμ1,μ3/m3bulk = -10 ± 1 m-1). Between these extremes, the magnitude of Γμ1,μ3/m3bulk decreases in the order glycine betaine » proline >TMAO > trehalose ≈ K+Glu- > glycerol. The order of exclusion of E. coli osmolytes from BSA surface correlates with their effectiveness as osmoprotectants, which increase the growth rate of E. coli at high external osmolality. For the most excluded solute (betaine), Γμ1,μ3 provides a min. est. of the hydration of native BSA of approx. 2.8 × 103 H2O/BSA, which corresponds to slightly less than a monolayer (estd. to be ∼3.2 × 103 H2O). Consequently, of the solutes investigated here, only betaine might be suitable for use in osmotic stress expts. in vitro as a direct probe to quantify changes in hydration of protein surface in biopolymer processes. More generally however, our results and anal. lead to the proposal that any of these solutes can be used to quantify changes in water-accessible surface area (ASA) in biopolymer processes once preferential interactions of the solute with biopolymer surface are properly taken into account.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXhvFWksL8%253D&md5=e5fa1534f3f1ba6d596387b362c98ece
65. 65
  Santoro, M. M., Liu, Y., Khan, S. M., Hou, L. X., and Bolen, D. W. (1992) Increased thermal stability of proteins in the presence of naturally occurring osmolytes. Biochemistry 31, 5278– 5283, DOI: 10.1021/bi00138a006
  65
  Increased thermal stability of proteins in the presence of naturally occurring osmolytes
  Santoro, Marcelo M.; Liu, Yufeng; Khan, Saber M. A.; Hou, Li Xiang; Bolen, D. W.
  Biochemistry (1992), 31 (23), 5278-83CODEN: BICHAW; ISSN:0006-2960.
  Organisms and cellular systems which have adapted to stresses such as high temp., desiccation, and urea-concg. environments have responded by concg. particular org. solutes known as osmolytes. These osmolytes are believed to confer protection to enzyme and other macromol. systems against such denaturing stresses. DSC expts. were performed on RNase A and hen egg white lysozyme in the presence of varying concns. of the osmolytes glycine, sarcosine, N,N-dimethylglycine, and betaine. Solns. contg. up to several molar concns. of these solutes resulted in considerable increases in the thermal unfolding transition temp. (Tm) for these proteins. DSC scans of RNase A in the presence of up to 8.2 M sarcosine resulted in reversible two-state unfolding transitions with Tm increases of up to 22° and unfolding enthalpy changes which were independent of Tm. On the basis of the thermodn. parameters obsd., 8.2 M sarcosine results in a stabilization free energy increase of 7.2 kcal/mol for RNase A at 65°. This translates into more than a 45,000-fold increase in stability of the native form of RNase A over that in the absence of sarcosine at this temp. Catalytic activity measurements in the presence of 4 M sarcosine give kcat and Km values that are largely unchanged from those in the absence of sarcosine. DSC of lysozyme unfolding in the presence of these osmolytes also results in Tm increases of up to 23°; however, significant irreversibility occurs with this protein. Naturally occurring glycine-based osmolytes appear to provide a general method of stabilizing proteins against thermal unfolding even well beyond the physiol. concn. range for osmolyte, and the degree of stabilization can be extraordinary.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XktVaqt70%253D&md5=fa1acd772b704d8c506795d8f7e64677
66. 66
  Tantos, A., Szrnka, K., Szabo, B., Bokor, M., Kamasa, P., Matus, P., Bekesi, A., Tompa, K., Han, K. H., and Tompa, P. (2013) Structural disorder and local order of hNopp140. Biochim. Biophys. Acta, Proteins Proteomics 1834, 342– 350, DOI: 10.1016/j.bbapap.2012.08.005
  66
  Structural disorder and local order of hNopp140
  Tantos, Agnes; Szrnka, Kriszta; Szabo, Beata; Bokor, Monika; Kamasa, Pawel; Matus, Peter; Bekesi, Angela; Tompa, Kalman; Han, Kyou-Hoon; Tompa, Peter
  Biochimica et Biophysica Acta, Proteins and Proteomics (2013), 1834 (1), 342-350CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)
  Human nucleolar phosphoprotein p140 (hNopp 140) is a highly phosphorylated protein inhibitor of casein kinase 2 (CK2). As in the case of many kinase-inhibitor systems, the inhibitor has been described to belong to the family of intrinsically disordered proteins (IDPs), which often utilize transient structural elements to bind their cognate enzyme. Here, the authors investigated the structural status of this protein both to provide distinct lines of evidence for its disorder and to point out its transient structure potentially involved in interactions and also its tendency to aggregate. The structural disorder of hNopp140 was apparent by its anomalous electrophoretic mobility, protease sensitivity, heat stability, hydrodynamic behavior on size-exclusion chromatog., 1H NMR spectrum, and DSC calorimetry scan. Phosphoprotein hNopp140 had a significant tendency to aggregate and the change of its CD spectrum in the presence of 0-80% trifluoroethanol suggested a tendency to form local helical structures. Wide-line NMR measurements suggested the overall disordered character of the protein. In all, the data suggested that this protein falls into the pre-molten globule state of IDPs, with a significant tendency to become ordered in the presence of its partner as demonstrated in the presence of transcription factor IIB (TFIIB).
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsV2qtLbI&md5=a69400cb1225796b669940c07d12fc11
67. 67
  van der Lee, R., Buljan, M., Lang, B., Weatheritt, R. J., Daughdrill, G. W., Dunker, A. K., Fuxreiter, M., Gough, J., Gsponer, J., Jones, D. T., Kim, P. M., Kriwacki, R. W., Oldfield, C. J., Pappu, R. V., Tompa, P., Uversky, V. N., Wright, P. E., and Babu, M. M. (2014) Classification of intrinsically disordered regions and proteins. Chem. Rev. 114, 6589– 6631, DOI: 10.1021/cr400525m
  67
  Classification of intrinsically disordered regions and proteins
  van der Lee, Robin; Buljan, Marija; Lang, Benjamin; Weatheritt, Robert J.; Daughdrill, Gary W.; Dunker, A. Keith; Fuxreiter, Monika; Gough, Julian; Gsponer, Joerg; Jones, David T.; Kim, Philip M.; Kriwacki, Richard W.; Oldfield, Christopher J.; Pappu, Rohit V.; Tompa, Peter; Uversky, Vladimir N.; Wright, Peter E.; Babu, M. Madan
  Chemical Reviews (Washington, DC, United States) (2014), 114 (13), 6589-6631CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
  A review. While many proteins need to adopt a well-defined structure to carry out their function, a large fraction of the proteome of any organism consists of polypeptide segments that are not likely to form a defined 3-dimensional structure, but are nevertheless functional. These protein segments are referred to as intrinsically disordered regions (IDRs). Proteins without IDRs are called structured proteins, and proteins with entirely disordered sequences that do not adopt any tertiary structure are referred to as intrinsically disordered proteins (IDPs). IDRs and IDPs are prevalent in eukaryotic genomes, with 44% of human protein-coding genes contg. disordered segments of >30 residues in length. Here, the authors discuss classification approaches based on function, functional elements, sequence, protein interactions, evolution, regulation, and biophys. properties. The authors also discuss resources that are currently available for gaining insight into IDR function, suggest areas where increased efforts are likely to advance the understanding of the functions of protein disorder, and speculate how combinations of multiple existing classification schemes could achieve high quality function prediction for IDRs, which should ultimately lead to improved function coverage and a deeper understanding of protein function.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXntFChur0%253D&md5=436cf71d9b77f6f3b176233a6abd9628
68. 68
  Uversky, V. N. (2013) The most important thing is the tail: multitudinous functionalities of intrinsically disordered protein termini. FEBS Lett. 587, 1891– 1901, DOI: 10.1016/j.febslet.2013.04.042
  There is no corresponding record for this reference.
69. 69
  Uversky, V. N. (2009) Intrinsically disordered proteins and their environment: effects of strong denaturants, temperature, pH, counter ions, membranes, binding partners, osmolytes, and macromolecular crowding. Protein J. 28, 305– 325, DOI: 10.1007/s10930-009-9201-4
  69
  Intrinsically disordered proteins and their environment: Effects of strong denaturants, temperature, pH, counter ions, membranes, binding partners, osmolytes, and macromolecular crowding
  Uversky, Vladimir N.
  Protein Journal (2009), 28 (7-8), 305-325CODEN: PJROAH; ISSN:1572-3887. (Springer)
  Av review. Intrinsically disordered proteins (IDPs) differ from "normal" ordered proteins at several levels, structural, functional and conformational. Amino acid biases characteristic for IDPs det. their structural variability and lack of rigid well-folded structure. This structural plasticity is necessary for the unique functional repertoire of IDPs, which is complementary to the catalytic activities of ordered proteins. Amino acid biases also drive atypical responses of IDPs to changes in their environment. The conformational behavior of IDPs is characterized by the low cooperativity (or the complete lack thereof) of denaturant-induced unfolding, lack of the measurable excess heat absorption peak(s) characteristic for the melting of ordered proteins, "turned out" response to heat and changes in pH, the ability to gain structure in the presence of various counter ions, osmolytes, membranes, and binding partners, and by the unique response to macromol. crowding. Here, the author describes some of the most characteristic features of IDP conformational behavior and the unique response of IDPs to changes in their environment.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtFGjt7jE&md5=84a0c51d56bf800bd65f0e3318a8c7d7
70. 70
  Abeln, S. and Frenkel, D. (2008) Disordered flanks prevent peptide aggregation. PLoS Comput. Biol. 4, e1000241 DOI: 10.1371/journal.pcbi.1000241
  There is no corresponding record for this reference.
71. 71
  Grana-Montes, R., Marinelli, P., Reverter, D., and Ventura, S. (2014) N-terminal protein tails act as aggregation protective entropic bristles: the SUMO case. Biomacromolecules 15, 1194– 1203, DOI: 10.1021/bm401776z
  71
  N-Terminal Protein Tails Act as Aggregation Protective Entropic Bristles: The SUMO Case
  Grana-Montes, Ricardo; Marinelli, Patrizia; Reverter, David; Ventura, Salvador
  Biomacromolecules (2014), 15 (4), 1194-1203CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
  The formation of β-sheet enriched amyloid fibrils constitutes the hallmark of many diseases but is also an intrinsic property of polypeptide chains in general, because the formation of compact globular proteins comes at the expense of an inherent sequential aggregation propensity. In this context, identification of strategies that enable proteins to remain functional and sol. in the cell has become a central issue in chem. biol. We show here, using human SUMO proteins as a model system, that the recurrent presence of disordered tails flanking globular domains might constitute yet another of these protective strategies. These short, disordered, and highly sol. protein segments would act as intramol. entropic bristles, reducing the overall protein intrinsic aggregation propensity and favoring thus the attainment and maintenance of functional conformations.
  >> More from SciFinder ^®
  https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXivFyqurs%253D&md5=d77448eec5800f23bda5fd4c25a812fb
72. 72
  Strader, M. B. (1998) Constructing a hybrid of R67 dihydrofolate reductase to study asymmetric mutations in the active site. M.S. Thesis, University of Tennessee, Knoxville, TN.
  There is no corresponding record for this reference.
73. 73
  Martinez, M. A., Pezo, V., Marliere, P., and Wain-Hobson, S. (1996) Exploring the functional robustness of an enzyme by in vitro evolution. EMBO J. 15, 1203– 1210
  There is no corresponding record for this reference.
Supporting Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00822.
- A 20% SDS–PAGE gel indicating clear separation of full-length and truncated R67 DHFRs, a flow diagram of our steps using MD and SASSIE to find N-terminal conformers that fit the SANS data, another figure depicting the SANS profile and GNOM analysis for truncated R67 DHFR as well as a dimensionless Kratky plot comparing full-length and truncated R67, the COM position for the N-terminal methionine of apo R67 DHFR conformers that fit the SANS data, SASSIE analysis of the data of the binary complex (R67 DHFR–NADP⁺), the ternary complex (R67 DHFR–NADP⁺–DHF), and R67 DHFR in the presence of 20% deuterated betaine, plots of R_g for full-length and truncated R67 DHFR probed after addition of betaine or DMSO, data mining plots for apo and ternary complex fits, and two tables listing R_g values from the various programs and T_M values from DSC data (PDF)
- Excel sheet from the data mining of apo conformers (XLSX)
- Excel sheet from the data mining of ternary conformers (XLSX)
Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N-TerminiClick to copy article linkArticle link copied!

Biochemistry

Abstract

Methods

Protein Expression and Purification

Small Angle Neutron Scattering (SANS)

Analysis Using MD and SASSIE

Data Mining

Differential Scanning Calorimetry (DSC)

Pressure Perturbation Calorimetry (PPC)

Results

SANS of Apo R67 DHFR

Figure 1

Figure 2

Effect of Ligand Binding on the Disordered Termini in R67 DHFR

Figure 3

Effect of Betaine on the R67 DHFR Structure

Figure 4

Osmolytes Probe Preferential Hydration of R67 DHFR

Figure 5

Effect of Osmolytes on the Thermal Stability of R67 DHFR

Figure 6

Pressure Perturbation Calorimetry

Discussion

Apoprotein Analysis

Figure 7

Analysis of Binary and Ternary Complexes

Effects of Osmolytes

Hydration Studies

Conclusion

Supporting Information

Terms & Conditions

Author Information

Acknowledgments

Abbreviations

References

Cited By

Biochemistry

Article Views

Altmetric

Citations

Recommended Articles

Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

References

Supporting Information

Supporting Information

Terms & Conditions

Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N-Termini
Click to copy article linkArticle link copied!