ACS Publications. Most Trusted. Most Cited. Most Read
An Unusually Rapid Protein Backbone Modification Stabilizes the Essential Bacterial Enzyme MurA
More

OR SEARCH CITATIONS

My Activity
Publications

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:Biochemistry 2020, 59, 39, 3683-3695
  • Open Access
Article

An Unusually Rapid Protein Backbone Modification Stabilizes the Essential Bacterial Enzyme MurA
Click to copy article linkArticle link copied!

Open PDFSupporting Information (1)

Biochemistry

Cite this: Biochemistry 2020, 59, 39, 3683–3695
Click to copy citationCitation copied!
https://doi.org/10.1021/acs.biochem.0c00502
Published September 15, 2020

Copyright © 2020 American Chemical Society. This publication is licensed under CC-BY.

Abstract

Click to copy section linkSection link copied!
High Resolution Image
Download MS PowerPoint Slide

Proteins are subject to spontaneous rearrangements of their backbones. Most prominently, asparagine and aspartate residues isomerize to their β-linked isomer, isoaspartate (isoAsp), on time scales ranging from days to centuries. Such modifications are typically considered “molecular wear-and-tear”, destroying protein function. However, the observation that some proteins, including the essential bacterial enzyme MurA, harbor stoichiometric amounts of isoAsp suggests that this modification can confer advantageous properties. Here, we demonstrate that nature exploits an isoAsp residue within a hairpin to stabilize MurA. We found that isoAsp formation in MurA is unusually rapid and critically dependent on folding status. Moreover, perturbation of the isoAsp-containing hairpin via site-directed mutagenesis causes aggregation of MurA variants. Structural mass spectrometry revealed that this effect is caused by local protein unfolding in MurA mutants. Our findings demonstrate that MurA evolved to “mature” via a spontaneous post-translational incorporation of a β-amino acid, which raises the possibility that isoAsp-containing hairpins may serve as a structural motif of biological importance.

Copyright © 2020 American Chemical Society

Subjects

what are subjects
Proteins are usually assembled from 20 different α-amino acids, linked via amide bonds in a sequence-specific manner. The polyamide backbone is often considered invariant and inert. Nevertheless, over typical protein lifetimes, asparagine (Asn) and aspartate (Asp) residues can spontaneously isomerize into isoaspartate (isoAsp) featuring an unusual β-peptidic linkage (Figure 1a). (1−3) This process is initiated by the nucleophilic attack of the (deprotonated) backbone amide nitrogen on the side chain amide or acid group in the case of Asn and Asp, respectively. The resulting succinimide intermediate hydrolyses with a slight preference (70–85%) in the α position, yielding a mixture of isoAsp and Asp residues. (1) In model peptides, this process occurs on time scales ranging from days to years, with Asn–Gly sequences rearranging most rapidly. (4) The rates are typically lower in proteins, especially for Asn residues that are embedded in secondary structure motifs or otherwise engaged in H-bonds involving the N–H of the i+1 residue. (5)

Figure 1

Figure 1. Formation of isoaspartate and occurrence in MurA. (a) Mechanism of the spontaneous formation and enzyme-catalyzed repair of isoaspartate (isoAsp). Deamidation of asparagine occurs by nucleophilic attack of the backbone nitrogen atom of the C-flanking amino acid. This process leads to formation of a metastable succinimide intermediate, which hydrolyses to a mixture of Asp and isoAsp linkages. isoAsp can be methylated by PIMT to promote succinimide formation and in turn revert some proportions to Asp (gray arrows). AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine. (b) The essential bacterial enzyme MurA features an isoAsp residue (black) in a hairpin, formed post-translationally from Asn67 (pdb: 1ejc). (3) (c) Sequence alignment of structurally characterized MurA variants. Residues that align with the solvent-facing residues of the β-sheet from the E. cloacae variant are shown in green. The isoAsp-containing hairpin is conserved in Enterobacteriaceae and Vibrionaceae. Alternative hairpin sequences are possible, albeit with a requirement for additional residues. Example β-hairpins with one (Bacillus anthracis, 3sg1, light blue (18)), two (Haemophilus influenzae, 2rl2, salmon (19)), and three (Streptococcus pneumoniae, 5wi5, purple (20)) additional residues were superimposed with the structure from E. cloacae (4e7b, black) and are shown on the right. β-Sheet residues are shown in gray, and side chains are omitted for clarity.

High Resolution Image
Download MS PowerPoint Slide
isoAsp formation is considered as protein damage and associated with several pathologies. (6) For example, isoAsp is thought to promote aggregation of Aβ in neurodegenerative diseases (7−9) and of eye lens crystallins (10) in cataract formation. To counteract the negative effects of isoAsp formation, nature has evolved a “repair” enzyme, protein l-isoaspartyl methyltransferase (PIMT). (11,12) PIMT-dependent methylation at the α-carboxylate promotes succinimide formation and subsequent hydrolysis into a mixture of Asp and isoAsp (Figure 1a). Successive repair cycles are thus required to fully convert isoAsp to Asp. The importance of PIMT is demonstrated by the observation that PIMT-knockout mice, which accumulate isoAsp in their proteome, exhibit many adverse phenotypes such as retarded growth, reduced lifespan, and seizures. (13,14)
Remarkably, the essential bacterial enzyme MurA has been shown to contain an isoAsp residue within a hairpin (Figure 1b), (3) although it is unknown whether this β-linked residue plays a functional role. MurA, a UDP-N-acetylglucosamine 1-carboxyvinyltransferase, is a cytosolic enzyme and antibiotic target that catalyzes the first committing step in cell wall biosynthesis. (15) A careful structural analysis by Eschenburg and Schönbrunn using X-ray crystallography has revealed that MurA from Enterobacter cloacae exhibits an isoAsp residue in an unusual β-hairpin. (3) isoAsp67, which arises from deamidation/rearrangement of Asn67, perfectly fits the electron density at this site (Figure 1b), whereas Asn is not accommodated by the electron density (Figure S1a) and exhibits unfavorable dihedral angles (Figure S1b,c). The sequence of the hairpin encompassing isoAsp67 is present in Enterobacteriaceae and Vibrionaceae (Figure 1c), where it forms an atom-efficient way to access this hairpin. In other structurally characterized MurA homologues, more residues are needed to provide a similar structure.
β-Peptidic linkages have been shown to provide unique structural features, primarily in the context of foldamers. (16,17) Such observations raise the possibility that isoAsp can provide access to unusual structural motifs and therefore confer favorable properties onto proteins. Here, we demonstrate that this is indeed the case for the naturally occurring isoAsp residue in MurA. We show that mutations precluding isoAsp formation at position 67 in MurA promote protein aggregation and negatively impact on MurA activity. Moreover, we found that isoAsp in MurA increases protein stability by rigidifying the movement of neighboring regions. Therefore, the conversion of Asn67 to isoAsp represents a desirable protein backbone “maturation” process, rather than molecular wear-and-tear which is commonly associated with isoAsp formation.

Methods

Click to copy section linkSection link copied!

Production and Purification of MurA

Plasmids encoding MurA variants were transformed into BL21(DE3) for protein production. 600 mL of LB containing 100 μg/mL ampicillin was inoculated with overnight cultures and grown at 37 °C with shaking at 220 rpm. When an OD600 of 0.5–0.8 was reached, protein production was induced with 1 mM IPTG, and the cultures were incubated for an additional 3.5–4 h at 37 °C with shaking. Cells were then pelleted by centrifugation (4200 rpm, 20 min, 4 °C). The pellets were resuspended in lysis buffer (50 mM Tris-HCl and 200 mM NaCl, pH 7.6) at room temperature, transferred to 50 mL conical tubes, and centrifuged again at 2211 rcf. The resulting cell pellets were stored at −20 °C.
Harvested cell pellets were resuspended in 20 mL of chilled lysis buffer and incubated for 30–60 min on ice in the presence of 1 mL of 10 mg/mL lysozyme, 20 μL of 1 M MgCl2, and 20 μL of 25 mg/mL DNase. Cells were lysed by sonication (25 cycles; 10 s pulse at 20% amplitude with 30 s off after each pulse) on ice, and the debris was pelleted at 15 000 rcf for 30 min. The supernatant was gently shaken with 2 mL of Ni-NTA resin (Qiagen) pre-equilibrated with lysis buffer at 4 °C for 30–60 min in a plastic column. Unbound proteins were removed, and the resin was washed with 15 mL of lysis buffer, followed by 15 mL of lysis buffer containing 10 mM imidazole and finally with 15 mL of lysis buffer containing 20 mM imidazole. The His-tagged MurA was eluted with lysis buffer containing 300 mM imidazole. Buffers and eluted fractions were kept on ice. Elution fractions were combined and concentrated to 0.5–1 mL using a Vivaspin concentrator (5 or 10 kDa MW cutoff). The MurA variants were further purified by size-exclusion chromatography (SEC) on an AKTA FPLC instrument equipped with a Superdex 200 10/300 GL SEC column. Samples were eluted with 1.5 column volumes of 50 mM Tris-HCl and 200 mM NaCl, pH 7.6, at 4 °C and a flow rate of 0.5 mL/min. Fractions containing monomeric MurA were pooled, concentrated, and stored at −80 °C.
The S200 column was calibrated with blue dextran and protein standards (Sigma-Aldrich) consisting of carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), amylase (200 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa).

Cleavage of the His-SUMO Tag

For biophysical characterization, the His-SUMO tag was cleaved as follows. His-SUMO-MurA was partially denatured with 2 M urea by adding an equal volume of chilled denaturing buffer (50 mM Tris, 100 mM NaCl, 4 M urea, 2 mM DTT, pH 7.6). Next, ULP1 protease was added at a ratio of 1:100 (protease: MurA, by mass). After gentle manual mixing, the cleavage reaction was incubated overnight at 4 °C. The next day, the reaction was mixed with Ni-NTA resin pre-equilibrated with Tris-urea buffer (50 mM Tris, 150 mM NaCl, 2 M urea, 1 mM DTT, pH 7.6) and shaken at 4 °C for 60 min. The flow-through containing MurA was collected, and the Ni-NTA resin was washed with 15 mL of Tris-urea buffer containing 10 and 20 mM imidazole, respectively. The flow-through and wash fractions were combined and dialyzed 2× against 1 L of 50 mM Tris, 200 mM NaCl, and 1 mM DTT at pH 7.6 at 4 °C. After dialysis, the protein solution was centrifuged at 15 000 rcf, 4 °C for 30 min. The supernatant was concentrated using a Vivaspin concentrator (5 or 10 kDa MW cutoff) to a final volume of 0.5–1 mL and subsequently purified on a Superdex 200 column as described above. The fractions corresponding to monomeric MurA were collected and analyzed on SDS-PAGE.

Quantification of isoAsp Content by PIMT Assay

isoAsp was quantified by measuring the amount of S-adenosyl-l-homocysteine (AdoHcy) generated from the PIMT-catalyzed methylation of isoAsp. (21) An AdoHcy standard curve was generated by injecting 60 μL of 0.25–5 μM AdoHcy onto an Agilent Zorbax 300SB-C18 column, eluting with a linear gradient of 0–30% acetonitrile (0.1% TFA) in water (0.1% TFA) at 50 °C, and recording the absorbance at 260 nm. A plasmid encoding His6-tagged human PIMT was obtained from Addgene (catalog no. 34852). The protein was produced and purified by Ni-NTA chromatography under standard conditions.
Initial attempts to quantify isoAsp in MurA (2 μM) under native conditions were performed with 0.73 μM human PIMT. Optimized conditions to detect inaccessible isoAsp included a preincubation step in 3 M GdmCl at room temperature for 30–60 min. Other reaction components were then added to final concentrations of 2 μM MurA; 0.5 M GdmCl; 10 μM AdoMet; 14.6 μM human PIMT in 0.1 M NaPhos pH 6.8; 1 mM EDTA; and 0.16% Triton. The reactions (100 μL total volume) were incubated for 30 min at 30 °C. Subsequently, the reaction was quenched by adding 20 μL of 0.3 M phosphoric acid. 60 μL of the quenched reaction was injected to the Agilent Zorbax 300SB-C18 column with a linear gradient of 0–30% acetonitrile at 50 °C. The amount of isoAsp in MurA variants was calculated from the absorbance of the AdoHcy peak at 260 nm based on an AdoHcy standard curve (Figure S2). All AdoHcy peak areas were corrected based on a reaction conducted in the absence of substrate. Control peptides were included to verify the activity and specificity of PIMT under the assay conditions (sequence: SDLWKLLPEXNVLSPLPSQA, where X corresponds to either isoAsp or Asp).

Peptide Mapping by LysC Digest

LysC was dissolved into water to a final concentration of 1 mg/mL. A 1 μL portion of the LysC solution was added to 20 μL of 25 μM MurA in 50 mM Tris-HCl and 200 mM NaCl, pH 7.6 in an Eppendorf tube. The mixture was incubated at 37 °C for 21 h. After the incubation, 5 μL of LC buffer C (water containing 0.1% formic acid) was added, and the total 26 μL sample was transferred to an LC-MS vial. A 10 μL portion (192 pmol) was injected for LC-MS analysis.
An LC-MS analysis was conducted on a Waters Acquity UPLC CSH C18 1.7um instrument, 2.1 × 150 mm at 55 °C with a flow rate of 0.4 mL/min. The solvent system is water +0.1% formic acid (solvent C)/acetonitrile +0.1% formic acid (solvent D). The total run time was 45 min: 0–1.0 min (2% D); 1.0–29.0 min (2–45% D); 29.0–34.0 min (45–60% D); 34.0–35.0 min (60–95% D); 35.0–40.0 min (95–2% D); and 40.0–45.0 min (2% D). The data were analyzed on UNIFI, and the coverage was 98% for MurA. The peaks corresponding to peptides encompassing residues 64–88 (VERXGSVWIDASNVNNFSAPYDLVK, where X is Asn, (iso)Asp, or a succinimide) were further analyzed by MSE fragmentation. The following mass spectrometer settings were used: positive polarity, capillary voltage 3.0 kV, source temperature 120 °C, sampling cone 60 V, desolvation temperature 350 °C, m/z range 50–3000, and transfer collision energy ramp 20–45 V.

Peptide Mapping by AspN Digest

AspN protease (New England Biolabs) was freshly reconstituted in pure water to a concentration of 1 mg/mL. A 1 μL portion of the AspN solution was added to 20 μL of 25 μM MurA in 50 mM Tris-HCl, 100 mM NaCl, 1 M urea, and 2.5 mM ZnSO4, pH 7.6 in a PCR tube. The mixture was incubated at 36 °C for 21 h. After the incubation, 5 μL of LC solvent C (water containing 0.1% formic acid) was added, and the total 26 μL sample was transferred to an LC-MS vial. A 5 μL portion was injected for LC-MS analysis. LC/MS and MSE were performed as described for LysC-based peptide mapping. Peptides were automatically detected with UNIFI and manually analyzed with MassLynx.

Measurement of the Rate of isoAsp Formation in MurA

To enable the measurement of the rate of isoAsp formation in vitro, MurA containing a high proportion of unmodified Asn67 was prepared (MurAWT*). For this purpose, the production and purification procedure described above was accelerated as follows. MurA was produced in 200 mL batches with IPTG induction at 37 °C for 40–60 min. After expression, the culture was pelleted by centrifugation, resuspended in lysis buffer, and lysed by sonication. The lysate was rapidly purified by Ni-NTA and size exclusion chromatography. The purified fractions from SEC were immediately aliquoted, flash-frozen with liquid N2, and stored at −80 °C. The entire purification was carried out at 4 °C within 5–6 h to minimize isoAsp formation.
To measure the rate of isoAsp formation in MurA under native conditions, MurAWT* (42.5 μM in 50 mM Tris-HCl and 200 mM NaCl, pH 7.6) was incubated at 37 °C in a PCR block, and 12 μL aliquots were removed at specified intervals. Samples were quenched by freezing and stored at −80 °C. The isoAsp content of all time points was measured in parallel using the PIMT assay under partially denaturing conditions as described above. To measure the rate of isoAsp formation in denatured MurA, MurAWT* (42.5 μM in 50 mM Tris-HCl and 200 mM NaCl, pH 7.6) was mixed with an equal volume of 6 M GdmCl to reach a final GdmCl concentration of 3 M. Aliquots of the mixture were incubated at 37 °C and analyzed as above. Of note, MurA contains 13 Asn sites, two of which are followed by a glycine (Asn67 and Asn148) which likely deamidate with similar rates under denaturing conditions. The data were fitted to the equation
The kinetics of isoAsp formation in the peptide MurA63–73 were determined by the PIMT assay as described above or using a direct RP-HPLC method. For the former, aliquots of HPLC-purified peptide were dissolved in 50 mM Tris-HCl and 200 mM NaCl, pH 7.6 buffer, to a concentration of 50 μM (determined by nanodrop), incubated at 37 °C, and analyzed by the PIMT assay described above. Alternatively, peptides were dissolved in PBS buffer at a concentration of approximately 375 μM and incubated at 48, 55, 65, or 75 °C. Aliquots were removed, and 20 μL was analyzed by RP-HPLC using a PLRP-S column with 15–40% B at 70 °C. The disappearance of the peak corresponding to the Asn-containing peptide was fitted to an exponential decay (GraphPad Prism), and the half-life at 37 °C was extrapolated using an Arrhenius plot.

MurA Kinetics Assay

MurA activity assays were performed in a 96-well plate at 37 °C for 10–15 min in a final volume of a 150 μL reaction mixture containing 50 mM Tris-HCl-pH 7.5, 10 mM NaCl, 1 mM dithiothreitol (DTT), 0.6 mM UDP-GlcNAc (Sigma), and 44 nM MurA variant. Phosphoenolpyruvate (1 mM, PEP, Sigma-Aldrich) was added to initiate the reaction. The amount of inorganic phosphate (Pi) released was quantified by Malachite Green assay reagent (made in house) following the commercial supplier’s protocol (Cayman Chemical Company). Briefly, 150 μL of the reaction mixture was quenched by the addition of 30 μL of reagent A. After the subsequent addition of 30 μL of reagent B, sample absorbance was measured at 620 nm using a multiwell plate reader. Absorbance was converted to [Pi] using a standard curve generated from phosphate standards. Initial velocities were calculated by linear regression. All rates were corrected for the background reaction in the absence of MurA.
To measure the Km for UDP-GlcNAc, the above enzymatic assay was performed with different concentrations of UDP-GlcNAc (0–400 μM) at a fixed concentration of 1 mM PEP. The average velocity (μmol Pi/min) at each UDP-GlcNAc concentration was determined, and Km,app(UDP-GlcNAc) and kcat,app values were obtained by fitting the data to the Michaelis–Menten equation in GraphPad Prism.

In Vitro Aggregation Assays

In vitro aggregation of MurA variants was induced by incubation at low or high ionic strength, high temperature, and/or low pH and monitored by analytical SEC. Stock solutions of 45 μM MurA in 50 mM Tris-HCl and 200 mM NaCl, pH 7.6, were diluted with four volumes of phosphate buffered saline (PBS) of varying compositions. Final conditions were as follows: 150 mM NaCl, 10 mM Tris, 8 mM sodium phosphate, 2 mM KCl, pH 7.5 (normal); 1.6 M NaCl, 10 mM Tris, 8 mM sodium phosphate, 2 mM KCl, pH 7.5 (high salt); 40 mM NaCl, 10 mM Tris, 8 mM sodium phosphate, 2 mM KCl, pH 7.5 (low salt); and 150 mM NaCl, 10 mM Tris, 8 mM sodium phosphate, 2 mM KCl, pH 6.0–6.2 (low pH). These samples were incubated as indicated, centrifuged, and injected onto an Agilent Advancebio SEC column (column volume 5 mL). Proteins were eluted with 50 mM Tris and 200 mM NaCl, pH 7.6, at a flow rate of 0.3 mL/min for 20 min. The recovery yield of proteins was calculated based on the area of monomeric MurA at a wavelength of 280 nm as compared to an untreated control sample. Measurements were conducted in triplicate.
To partially denature samples, MurA stock solutions in 50 mM Tris-HCl and 200 mM NaCl, pH 7.6, were diluted with an equal volume of 50 mM Tris-HCl, 100 mM NaCl, 2 mM DTT, and 4 M urea. Samples in 2 M urea were incubated for 3 h at room temperature and then dialyzed in 1 L of 50 mM Tris-HCl, 200 mM NaCl, and 1 mM DTT, pH 7.6, overnight at 4 °C using a dialysis button. The amount of aggregation was estimated also by analytical size-exclusion chromatography.

CD Spectroscopy

Far-UV CD spectra of protein samples were recorded at 25 °C on an Aviv 202 spectrometer from 270 to 200 nm in 1 nm steps with an averaging time of 1 s in 10 mM sodium phosphate and 150 mM NaCl, pH 7.6. MurA concentrations were 3.0 μM (1 mm cuvette) or 30 μM (0.1 mm cuvette). Two scans were averaged, and a buffer blank was subtracted.
For chemical denaturation, the denaturation curve was obtained by measuring ellipticity of a 3 μM protein solution at 222 nm using a 1 mm cuvette, at urea concentrations between 0 and 9 M in 10 mM phosphate and 150 mM NaCl, pH 7.6 at 25 °C. The signal of each point was averaged over 1 min. Each urea concentration was diluted from a 10 M urea, 10 mM phosphate, and 150 mM NaCl, pH 7.6 stock solution individually, and the CD signal was measured after a 15 min equilibration time at 25 °C. The raw data from Aviv 202 software were fitted nonlinearly to a two-state unfolding model with baseline correction using GraphPad Prism:
where αN and αD are the CD signals of the native and denatured states, respectively. βN and βD are the slopes at low and high urea concentrations, respectively. Y is the raw CD signal, mD–N the cooperativity of unfolding, and D50% the denaturant concentration at which 50% of protein is unfolded.
Reversibility of chemical denaturation was assessed by measuring the kinetics of MurA denaturation and refolding. A 3 mL cuvette was used, and the protein concentration was 0.3 μM. Denaturation kinetics were measured at 25 °C immediately after diluting a protein stock to 3 mL with urea buffer (2 M urea concentration); refolding kinetics were measured at 25 °C after incubating the protein in 300 μL of 9 M urea buffer for 15 min at 25 °C and quickly diluting the solution to 2 M urea. Denaturation and refolding signals converged over time for both MurAWT and MurAN67D. Under these conditions, refolding was >85% reversible for both variants (Figure S3).
A thermal denaturation curve was obtained by measuring ellipticity of a 1 μM protein solution at 222 nm between 4 and 95 °C. The sample was heated in 1 °C steps in 10 mM phosphate and 150 mM NaCl, pH 7.6, with a 1 min equilibration time and a 1 min signal averaging time per point. A 10 mm CD cuvette equipped with a stir bar was used (total volume 3 mL). The fraction of folded MurA was calculated under the assumption that the unfolded fraction of the protein is 0 and 1 at 4 and 95 °C, respectively. An additional correction for temperature-specific change in CD signal was taken into account as follows. The data points from 4 to 30 °C and from 80 to 95 °C were fitted to a linear equation and extrapolated to reflect the folded θF(T) and unfolded θU(T) signal as a function of temperature. The fraction of unfolded protein at each temperature (FU) is then calculated from the signal θ using the following equation:
The melting temperatures were approximated through fitting the data to a 3-state model in GraphPad Prism using the following equation:
where FI is the contribution of the unfolding intermediate to the CD signal, R is the universal gas constant, Tm1 and Tm2 are the melting temperatures for the first and second transition, and ΔHm1 and ΔHm2 are the enthalpy of denaturation at the respective transition midpoint. A representative trace from 2 independent measurements is shown.
In parallel, midpoints of transition were also determined from maxima of the first derivative of the thermal denaturation curve (after smoothing with a second degree polynomial and 4 adjacent data points) in GraphPad Prism.

Analysis of MurA Flexibility and Solvent Accessibility via HDX-MS

HDX-MS experiments (22) were carried out on a Synapt G2Si (Waters, UK) instrument coupled with a commercial HDX system (Waters, UK) and Acquity M-Class system. The instrument was calibrated with sodium iodide and run in sensitivity mode and MSE fragmentation mode with electrospray ionization. The following mass spectrometer settings were used: positive polarity, capillary voltage 3.0 kV, source temperature 80 °C, sampling cone 30 V, desolvation temperature 200 °C, m/z range 50–2000, and transfer collision energy ramp 25–45 V. Leucine enkephalin (Waters, UK) was used as the lockmass.
Stock solutions of MurAWT and MurAN67D were prepared at 20 μM. A 5 μL sample was diluted with 95 μL of Buffer L (50 mM Tris-HCl and 200 mM NaCl in deuterium oxide, pH 7.6) to initiate the HDX reaction and incubated for either 0.25, 1, 30, or 120 min in triplicate. A 5 μL sample was diluted with 95 μL of Buffer E (50 mM Tris-HCl and 200 mM NaCl, pH 7.6) for undeuterated references for 6 replicates. A 70 μL portion of the reaction mixture was quenched with 70 μL of quench solution (100 mM potassium phosphate, pH 2.3) at 1 °C. A 100 μL portion of quenched sample was then injected onto the HDX system. Samples were digested online with a Waters Enzymate BEH pepsin column at 20 °C and trapped on a Waters BEH C18 VanGuard column at 0 °C. Digestion and trapping were carried out for 3 min at 200 μL/min with 100% Mobile Phase A (0.2% formic acid). Peptides were eluted from the trapping column and separated on a Waters BEH C18 column (100 mm × 1.0 mm) at 0 °C over a 7 min gradient of 8–40% Mobile Phase B (0.2% formic acid in acetonitrile) at 40 μL/min. All sample injections were followed with a clean blank with a sawtooth gradient to prevent carryover. The pepsin column was cleaned with pepsin wash (1.5 M GdmCl, 4% acetonitrile, and 0.8% formic acid) after each injection.
Peptides were identified using Waters ProteinLynx Global Server 2.5.1 (PLGS). Identified peptides were filtered and curated using Waters DynamX V3.0. Statistical analysis was performed using Deuteros v1.0 (King’s College London, UK) (23) using a global confidence interval of 99%. Peptides were not corrected for back-exchange as absolute uptake is not required for differential HDX. Biological replicates were carried out to confirm the HDX results.

Results

Click to copy section linkSection link copied!

MurA Features Stoichiometric Levels of isoAsp

Because of its biological importance (24) and the wealth of structural information, (3) MurA represents an ideal test case to dissect potentially favorable roles of isoAsp in protein structure and function. To verify the presence and extent of the isoAsp modification in E. cloacae MurA, we produced the enzyme in Escherichia coli as an N-terminal fusion with a hexahistidine-SUMO tag (SUMO-MurAWT). The resulting protein was purified by Ni-NTA affinity chromatography and gel filtration. The main peak in the size exclusion chromatogram corresponds to a monomer (Figure S4a; observed MW, 60 kDa; expected MW, 57 kDa). Approximately 10% of the protein eluted in the column void volume, presumably in the form of soluble aggregates. The monomer fraction contains pure MurA as judged by SDS PAGE (Figure S5), and the identity of the protein was confirmed by mass spectrometry (Figure S6a). To measure the extent of isoAsp modification in our preparation of SUMO-MurAWT, we used a previously described enzymatic assay. (25) PIMT selectively methylates isoAsp using the cofactor S-adenosyl methionine (AdoMet), concomitantly forming S-adenosyl-l-homocysteine (AdoHcy) which is quantified by reverse phase (RP) HPLC. To our surprise, we observed only 3.5 ± 1.9 pmol of isoAsp per 100 pmol of SUMO-MurAWT when the assay was performed under native conditions using a catalytic amount of PIMT (Figure 2a). Under the same conditions, control peptides bearing an isoAsp or an Asp residue exhibited 93 ± 12 pmol and 0.5 ± 0.1 pmol of isoAsp, respectively (Figure 2a and Figure S4b). Given that the relatively narrow active site of PIMT (26) is unlikely to accommodate folded proteins, we denatured MurA with 3 M GdmCl prior to the PIMT assay and increased the concentration of PIMT during the assay. Under these conditions, stoichiometric amounts of isoAsp were detected (98 ± 3 pmol, Figure 2a, Figure S4c). The near complete conversion to isoAsp as well as the requirement for MurA denaturation indicate that the isoAsp is embedded in a structural motif, consistent with isoAsp67 in several crystal structures. (3,27) We speculate that the MurA fold also prevents access of PIMT to isoAsp67 in vivo.

Figure 2

Figure 2. SUMO-MurAWT contains stoichiometric amounts of isoAsp. (a) Biochemical determination of SUMO-MurAWT isoAsp content via PIMT-dependent methylation. 100 pmol of SUMO-MurAWT or control peptides with or without an isoAsp residue is used as the substrate. AdoHcy is quantified by RP-HPLC upon methylation of SUMO-MurAWT under native conditions using catalytic amounts of PIMT (0.73 μM, red) or after preincubation with 3 M GdmCl and treatment with 14.6 μM PIMT (blue). (b–f) Asn67 is fully deamidated: (b) LC-MS analysis of LysC-digested MurAWT. The peaks at 14.7 (c), 15.0 (d), and 15.4 (e) min correspond to peptide VERXGSVWIDASNVNNFSAPYDLVK (residues 64–88 in MurA), with X being Asn, succinimide, and Asp and isoAsp, respectively. The dashed line in part e represents the scale of the spectra in parts c and d. (f) MSE analysis of the 15.4 min peak from part e. The masses of ions B3 (calcd, 385.22 Da; observed, 385.22 Da) and B4 (calcd, 500.25 Da; observed, 500.25 Da) confirm that X corresponds to an (iso)Asp residue.

High Resolution Image
Download MS PowerPoint Slide
To further confirm the site of isoAsp formation, we performed LC-MS analysis of LysC-digested MurAWT (Figure 2b). We detected several isoforms of the peptide corresponding to residues 64–88 (Figure 2c–e). Among these, the most intense peak (retention time 15.4 min, Figure 2e) exhibited a mass difference of +1 Da from the encoded sequence, indicating that, as expected, deamidation occurred. Based on the high isoAsp content determined in the PIMT assay, we assign the 15.4 min peak to the isoAsp67 form. Masses corresponding to the unmodified Asn sequence (retention time 14.7 min, Figure 2c) and a succinimide isoform (retention time 15.0 min, Figure 2d) were also present, albeit at 60-fold lower intensities. Fragmentation of the deamidated peak by MSE analysis further confirmed the location of the deamidation at residue 67 (Figure 2f). Collectively, these results underscore that Asn67 has post-translationally isomerized to isoAsp to an extent of >95% in our preparations of MurAWT.

The Tertiary Structure of MurA Promotes Rapid isoAsp Formation

The presence of almost stoichiometric amounts of isoAsp at position 67 suggests that its formation is accelerated in MurA. We therefore sought to determine the rate of isomerization of this site in vitro. To access Asn67-containing MurA, we performed a rapid protein expression (1 h at 37 °C) and purification (∼5 h at 4 °C) of SUMO-MurAWT. PIMT assays of flash-frozen aliquots from this preparation (MurAWT*) revealed an isoAsp content of 22%, thus enabling a measurement of the rate of isoAsp formation. At 37 °C, MurAWT* isomerizes with a half-life of 4.7 h, yielding >95% isoAsp content after 24 h (Figure 3). This rate is considerably faster than values for deamidation of Asn–Gly sequences measured in peptides (half-life ≈ 1 day) (4) and estimated in proteins (mean half-life ≈ 100 days). (28) Given the unusually high rate of isoAsp formation, we explored whether the structure of MurA facilitates this transformation. We incubated MurAWT* at 37 °C in the presence of 3 M GdmCl to denature the protein. Under these conditions, isoAsp formation was >10× slower than under native conditions (half-life ≈ 80 h, Figure 3), which suggests that isoAsp formation in MurA is autocatalytic. Notably, the curve plateaus at approximately 200% isoAsp content under denaturing conditions. This observation is consistent with the presence of two Asn–Gly sequences in MurA (Asn67 and Asn148), which are expected to isomerize with similar rates in the unfolded protein. A synthetic peptide encompassing residues 63–73 (MurA63–73) also isomerizes with a half-life ≈60 h (Figure 3 and Figure S7), an order of magnitude slower than folded MurA. To further confirm this result, we determined the deamidation kinetics of the peptide MurA63–73 at elevated temperatures directly by HPLC (Figure S8). An Arrhenius plot shows that the temperature dependence of the deamidation rate is linear, allowing extrapolation to 37 °C (Figure 3c). The resulting half-life of ≈70 h is in good agreement with measurements using the PIMT assay (Figure 3d), further supporting the premise that the 3-dimensional structure of MurA is crucial for rapid isoAsp formation.

Figure 3

Figure 3. The 3D-structure of MurA facilitates Asn isomerization. (a) Measurement of the isoAsp content of MurAWT* after maturation at 37 °C under native (blue circles) and denaturing (3 M GdmCl, green triangles) conditions. Data points for MurAWT* under native conditions are from two independent measurements. MurA contains two Asn–Gly sequences, expected to isomerize with similar rates under denaturing conditions. The isoAsp content of a peptide corresponding to residues 63–73 (MurA63–73) is shown in pink diamonds. (b) Zoom-in for the initial 24 h period in part a (shaded in gray). (c) Temperature dependence of MurA63–73 deamidation measured by HPLC at 48, 55, 65, and 75 °C. An Arrhenius plot is shown in the inset. (d) Half-lives for isoAsp formation at 37 °C. Error bars represent the 95% CI of the fit from parts a and c.

High Resolution Image
Download MS PowerPoint Slide

isoAsp67 Improves the Yield of Recombinant MurA

The accelerated formation of stoichiometric isoAsp in MurA suggests that this backbone modification serves an important purpose. To address this hypothesis, we generated a series of MurA mutants and analyzed their properties. An Asn67Asp (MurAN67D) mutation diminishes isoAsp formation at physiological pH and allows a direct comparison between two variants with identical functional groups but distinct backbone connectivity. Mutations of the flanking residues Arg66 and Gly68 to Ala (MurAR66A and MurAG68A, respectively) were introduced to probe their role in the isoAsp-containing β-hairpin. Arg66 contributes a salt bridge to the isoAsp α-carboxylate. The sterically minimal Gly68 is beneficial for rapid isoAsp formation; its mutation is expected to diminish isoAsp formation. All variants were produced in E. coli as His6-SUMO fusions and purified by Ni-NTA chromatography and gel filtration. Strikingly, the mutants all exhibit considerable amounts of soluble aggregates and, accordingly, reduced yields of monomeric MurA (Figure 4a). This observation establishes that the isoAsp-containing hairpin is an important structural feature of MurA.

Figure 4

Figure 4. Characterization of MurA variants. (a) Monomer fraction (gray, left axis) and yield (red, right axis) for SUMO-MurA variants after production and purification by Ni-NTA and size-exclusion chromatography (error bars: s.d. of four (WT) or three (mutants) biological replicates). (b) isoAsp-quantification for SUMO-MurA variants (error bars: s.d. of three biological replicates). The AdoHcy signals were corrected for the AdoHcy signal observed in a control reaction in the absence of substrate. (c) Relative activity measurement of SUMO-MurA variants (error bars: s.d. of four independent experiments). (d) Saturation kinetics of selected variants (error bars: s.d. of four independent experiments). Activities were recorded with 44 nM MurA variant, 1 mM PEP, and 0.6 mM UDP-GlcNAc unless otherwise stated.

High Resolution Image
Download MS PowerPoint Slide
We isolated the monomeric fractions of the MurA mutants (see Figures S5 and S6 for SDS-PAGE and mass spectra, respectively) and determined their isoAsp content (Figure 4b). SUMO-MurAN67D exhibited <5% isoAsp which confirms that position 67 is indeed the predominant site of MurA isomerization. We further verified the modification state of residue 67 by peptide mapping. LC-MS analysis of LysC-digested MurAWT and MurAN67D revealed for each variant a predominant peak corresponding to residues 64–88 based on mass (Figure S9). Importantly, these isobaric peptides exhibited distinct retention times, as confirmed by coinjection (Figure S9c). Based on the total ion count for these peaks, we estimate that MurAWT contains >90% isoAsp. Conversely, MurAN67D exhibits predominantly Asp with approximately 10% isoAsp, slightly higher than that determined via the PIMT assay. We did not detect additional isobaric peaks that could reflect the stereoisomers d-isoAsp and d-Asp, although their presence in small amounts cannot be excluded. To further validate this assignment, we performed a peptide mapping experiment with Asp-N (Figure S10) which cleaves N-terminal to Asp residues but cannot cleave isoAsp. (29) In MurAWT, a peptide encompassing residues 51–72 was observed with a retention time of 13.5 min and a mass of 2490.3 Da (Figure S10a,c). The signal for the same peptide was >10-fold lower in MurAN67D (Figure S10b,f). By contrast, high-intensity peaks for peptides originating from cleavage at Asp67 (residues 51–66, RT = 15.2 min, M + H+ = 1834.0 Da; and 67–72, RT = 13.6 min, M + H+ = 676.3 Da) were observed in the mutant (Figure S10g,h). These peaks were >100-fold lower in the wild type (Figure S10d,e), further supporting the premise that residue 67 of the wild type and the mutant is mostly isoAsp and Asp, respectively. As expected, (4) the Gly to Ala mutation decelerates isoAsp formation in SUMO-MurAG68A, with <30% of Asn67 being modified (Figure 4b). The MurAR66A mutant retained the ability to rapidly form isoAsp, demonstrating that Arg66 is not essential for this process.
We subsequently turned our attention to the catalytic activities of MurA variants. We quantified the phosphate released during MurA-catalyzed transfer of the enolpyruvyl moiety of PEP to UDP-GlcNAc. (30) Measurements of the initial velocities of MurA variants indicated that the activity of all mutant MurA were lower than the activity of the wild type, albeit only slightly (Figure 4c). The modest reduction in activity suggests that all variants retain the native MurA structure overall and is furthermore consistent with the location of isoAsp67 distal from the enzyme active site. To gain more insight into the nature of the reduction of activity, apparent Km,app(UDP-GlcNAc) and kcat,app values of SUMO-MurAWT and SUMO-MurAN67D were determined at a constant concentration of PEP (1 mM) (Figure 4d). The Km,app(UDP-GlcNAc) values were indistinguishable (97 μM and 103 μM for the isoAsp- and the Asp-containing variants, respectively, Table 1). By contrast, the kcat,app values differed by 20–25%, with SUMO-MurAWT exhibiting the higher maximum velocity compared with the N67D mutant (Table 1). Thus, the lower activity of SUMO-MurAN67D is likely due to a reduced proportion of active enzyme, although subtle changes to the active site cannot be excluded.
Table 1. Kinetic Characterization of MurA Variants
variantarelative activitya (μM/min)kcat,appa (s–1)Km,app(UDP-GlcNAc)a (μM)kcat,app/Km,app (mM–1 s–1)
SUMO-MurAWT4.4 ± 0.33.9 ± 0.1997 ± 1340 ± 8
SUMO-MurAN67D3.7 ± 0.33.0 ± 0.15103 ± 1429 ± 6
SUMO-MurAR66A2.2 ± 0.2   
SUMO-MurAG68A3.0 ± 0.3   
a

Ranges represent the errors of the fit from data shown in Figure 4.

isoAsp67 Reduces Aggregation of MurA under Stress Conditions

Given that MurA variants with mutations in the isoAsp loop readily aggregate during protein production or purification from E. coli lysate, we sought to investigate MurA aggregation vulnerability in a more controlled way in vitro. We performed these measurements with MurAWT and MurAN67D upon removal of the His-SUMO tag with Ulp1 protease, followed by size-exclusion chromatography. The cleaved variants were judged as pure by SDS-PAGE and mass spectrometry (Figures S5, S11, and S12). Circular dichroism spectroscopy and native ion mobility mass spectrometry indicate that the MurAWT and MurAN67D mutant have a similar secondary structure and overall shape (Figure S13), confirming that their folds are the same.
The two variants were subjected to various stress conditions and their quaternary structures analyzed by size exclusion chromatography. Specific challenges included high temperature (42 and 47 °C vs 37 °C), low pH (pH 6.0 vs 7.5), extreme salt concentrations (1.6 M and 40 mM vs 150 mM NaCl), and combinations thereof. Of note, for both MurA variants, the isoelectric point is pI = 5.7. After incubation at 37 °C overnight, both variants retained their monomeric state when exposed to low and high concentrations of NaCl or low pH (Figure 5a, Figure S14a,b). Upon treatment at 42 °C overnight or 47 °C for 1 h, however, MurAN67D aggregated under otherwise ambient conditions (45 ± 5% monomeric, Figure 5a, Figure S14c,d). Aggregation was exacerbated under low salt conditions (22 ± 3% monomeric) as well as at low pH (<10% monomeric) but diminished in the presence of 1.6 M NaCl. The isoAsp linkage clearly provides a protective effect under heat shock treatment, with more than 50% of MurAWT retaining its monomeric form under all conditions tested (Figure 5a). Partial denaturation with 2 M urea for 3 h, followed by renaturation by dialysis into native buffer, also resulted in the formation of approximately 80% aggregate for MurAN67D and only 20% for MurAWT (Figure 5b, Figure S14e). Collectively, these results suggest that the unusual isoAsp-containing hairpin plays an important role in maintaining the structural integrity of Enterobacterial MurA under stress conditions.

Figure 5

Figure 5. isoAsp formation improves MurA resilience against stress conditions. (a) Relative recovery of monomeric MurAWT and MurAN67D after incubation at nonoptimal temperatures, pH, and salt concentrations. Standard conditions: 150 mM NaCl, 10 mM Tris, 8 mM sodium phosphate, 2 mM KCl, pH 7.5. High salt: standard conditions with 1.6 M NaCl. Low salt: standard conditions with 40 mM NaCl. Low pH: standard conditions with pH = 6.0–6.2 (error bars: s.d. of three independent experiments). (b) Refolding yield upon partial denaturation with urea. MurAWT and MurAN67D were incubated in 2 M urea for 3 h at room temperature and subsequently dialyzed overnight at 4 °C.

High Resolution Image
Download MS PowerPoint Slide
The stability of MurA variants was further explored by chemical and thermal denaturation. Circular dichroism spectroscopy (CD) revealed that both variants unfold cooperatively in the presence of urea (Figure 6a, Figure S3). Based on a 2-state model, (31) the urea concentrations required to unfold 50% of MurAWT and MurAN67D are 2.6 and 2.0 M, respectively. These values indicate that MurAWT is more resilient against chemical denaturation. However, the different cooperativities of unfolding (mD–N = 5.1 ± 0.3 and 8.6 ± 0.9 kJ/(mol M) for MurAWT and MurAN67D, respectively) preclude a quantitative comparison of the free energy of unfolding. Thermal denaturation experiments confirmed that MurAWT unfolds cooperatively with an apparent midpoint of thermal unfolding at 58.5 °C (Figure 6b). Inspection of the first derivative of the thermal unfolding curve suggests the presence of an unfolding intermediate, indicated by a small shoulder at 53.0 °C (Figure 6c). This intermediate is much more pronounced for MurAN67D, which clearly exhibits two distinct transitions at 49.5 and 60.5 °C (Figure 6b,c). We thus conclude that isoAsp67 stabilizes MurA by preventing partial unfolding at lower temperatures.

Figure 6

Figure 6. isoAsp67 impacts the stability of MurA. (a) Chemical denaturation of MurAWT (blue squares) and MurAN67D (green triangles). The fraction unfolded was calculated from the circular dichroism signal at 222 nm. CD signals were recorded in the presence of 0–9 M urea at a protein concentration of 3 μM at 25 °C in a 1 mm cuvette. Unfolding curves are fitted to a two-state model as described in the methods section. (32) Individual data points from three (MurAWT) and four (MurAN67D) experiments are shown after correction for baseline slopes and calculation of the fraction unfolded at each [urea]. (b) Thermal denaturation of MurAWT (blue squares) and MurAN67D (green triangles). A representative plot from two independent measurements is shown. CD signals at 222 nm were recorded at a protein concentration of 1 μM between 4 and 95 °C at 1 °C intervals using a 1 cm cuvette. (c) Slope of the thermal denaturation curve. The first derivative from the curves in part b were calculated after smoothing the denaturation curve with a 2nd degree polynomial and 4 adjacent data points.

High Resolution Image
Download MS PowerPoint Slide

Mutation of isoAsp67 Causes Local Unfolding

We sought to gain structural insights into how isoAsp protects MurA from partial unfolding and aggregation with hydrogen–deuterium exchange mass spectrometry (HDX-MS). The technique measures the solvent exchange rate of backbone amide hydrogens in proteins and thereby provides a measure of local structural changes and protein dynamics. (33,34) Here, we used HDX, followed by on-column pepsin digestion and LC-MS (35) to measure the dynamics of MurAWT and MurAN67D. We compared differential exchange regimes ranging from 0.25 min to 2 h. Peptides within the regions of residues ∼40–55 and ∼55–70 exhibited increased deuterium uptake in the mutant MurAN67D as compared to MurAWT (Figure 7a,b, Figure S15). Such local changes in dynamics for the latter (residues ∼55–70, Figure 7d) were expected given that this region encompasses the isoAsp67-containing hairpin in MurAWT. The preceding region (∼40–55, Figure 7c), which also becomes more dynamic upon mutation of isoAsp67, corresponds to an amphiphilic helix and a connected loop which pack against the isoAsp-containing hairpin toward the protein interior. Thus, the HDX-MS experiments demonstrate that altering the unique connectivity of isoAsp through an N67D mutation increases local unfolding of the hairpin which propagates to neighboring structural elements on a time scale of minutes to hours.

Figure 7

Figure 7. HDX-MS analysis of MurA structure and dynamics. (a) Difference of deuterium uptake between MurAN67D and MurAWT. Woods plots (exposure time sums) from two biological replicates are combined. The dashed line corresponds to a 99% confidence interval. Mutation of isoAsp67 significantly increases the deuterium uptake between residues 41–55 and 55–71. (b) Regions of MurA displaying significantly enhanced deuterium uptake in MurAN67D as compared to MurAWT are colored in red and mapped onto the structure of MurA (pdb: 1ejc). (c–e) Deuterium uptake plots of peptides surrounding residues 67 of MurAWT (blue squares) and MurAN67D (green triangles). The location of the corresponding peptides within the structure of MurA is shown in gold (right panels). Two biological replicates were recorded (solid and dashed lines) with three technical replicates each (error bars: s.d.).

High Resolution Image
Download MS PowerPoint Slide

Discussion

Click to copy section linkSection link copied!
Post-translational backbone modifications are pervasive and play an important yet underappreciated role in protein biochemistry. (36) The rearrangement of Asn residues to β-Asp linkages is widely acknowledged to be involved in aging and damage of proteins and is thought to be the culprit for biological abnormalities. (8,13,14) Furthermore, the formation of isoAsp has been found to cause adverse immune reactions (37) and negatively impact therapeutic proteins (38) including monoclonal antibodies. (39,40) In stark contrast to such adverse effects, our work shows that the spontaneous formation of isoAsp in the β-hairpin of MurA from Enterobacter cloacae is beneficial. Remarkably, isoAsp formation was accelerated by an order of magnitude in folded MurA and yields isoAsp in an unusually homogeneous fashion. These observations suggest that the 3D structure of MurA enhances the formation of the succinimide intermediate as well as its regioselective hydrolysis, which supports the notion that MurA has evolved to exploit this residue. In agreement with this hypothesis, we found that perturbation of the isoAsp-containing hairpin by mutagenesis led to extensive aggregation during production and purification as well as under controlled stress conditions in vitro. Through biophysical analysis via HDX-MS we have pinpointed that replacement of the isoAsp with Asp causes local changes in dynamics and destabilizes nearby structural elements. Such local unfolding likely reveals hydrophobic surfaces which promote aggregation.
To understand the potential structural role of isoAsp, it is instructive to compare the hairpin of MurA to a typical Type II′ β-hairpin (41) (Figure 8a). Because Arg66 (φ = −137°, ψ = 172°) and Ser69 (φ = −103°, ψ = 167°) adopt a conformation compatible with β-sheet formation, we consider them as flanking the hairpin. In terms of position and conformation (φ = −67°, ψ = −21°), Gly68 takes the place of hairpin residue i + 2. isoAsp67 spans the distance covered by hairpin residues i and i + 1. An H-bond between the isoAsp backbone NH and the C═O of Ser69 (3.1 Å from N to O) results in a 12-membered H-bonded ring. In Type II′ β-hairpins, the similarly placed NH of residue i and C═O of i + 3 form a 14-membered H-bonded ring. The additional methylene group within the backbone of isoAsp enables this extra range at a minimal entropic cost given that the dihedral angle θ is 62° (N—Cα—Cβ—CO), a value that is almost ideal for the sp3–sp3 centers present in β-amino acids. (16,42) Another unique feature of isoAsp is the position of the α-carboxy group, which points in a direction otherwise only accessible to d-amino acids. This arrangement enables a salt bridge between isoAsp67 and Lys46 (Figure 8b). Indeed, our HDX-MS results are consistent with such an interaction, where the isoAsp-containing hairpin serves as a “lid” to stabilize a helix adjacent to Lys46.

Figure 8

Figure 8. isoAsp enables the formation of an unusual β-hairpin. (a) Comparison of the isoAsp-containing β-hairpin of MurA from E. cloacae (4e7b, yellow) with a typical Type II′ turn (1uxa, green). H-bonds are indicated by dashed lines. The backbone atoms of a 12- and 14-membered H-bonded ring are indicated for MurA (NH of isoAsp67 to CO of Ser69) and the Type II′ turn (NH of i to CO of i + 3), respectively. A 10-membered H-bonded ring of the Type II′ turn (CO of i to NH of i + 3) is shown in gray. ChemDraw structures of the turns are shown on the right (MurA, top; Type II′, bottom). (b) The α-carboxylate of isoAsp67 interacts with Lys46 (dashed line, O–N distance = 3.35 Å).

High Resolution Image
Download MS PowerPoint Slide
Investigations on the effect of artificially introduced β-amino acids have established that expansion of the peptide backbone by an additional methylene group is tolerated in many structural contexts, (43−45) including helices, (46) sheets, (47) turns, (48−50) and even within an enzyme active site. (51) In most cases, such synthetic backbone expansions lead to small losses in stability. The judicious choice of the site and residue can, however, result in hairpins which improve the stability of select proteins, for example RNase A (49) and the Pin1 WW domain. (52) The configurations of these artificial hairpins are distinct from the naturally occurring hairpin of MurA; whether the latter structure could similarly stabilize other proteins remains unexplored.
Other spontaneous protein backbone modifications have previously been shown to be beneficial for protein structure and function. The most prominent example is the fluorescent protein GFP from the jellyfish Aequorea victoria. In the process of establishing the fluorophore core of GFP, the three amino acids Thr–Tyr–Gly will undergo a series of backbone PTMs, including backbone cyclization, dehydration, and oxidation. (53) The methylene imidazolone (MIO) cofactor of amino mutases is established in a similar manner. (54) More recently, glutaminase from the hyperthermophilic archaeon Methanocaldococcus jannaschii has been shown to contain a stable succinimide, shielded from hydrolysis by surrounding residues. (55) This unusual residue contributes to the extraordinary stability of glutaminase by rigidifying its surroundings. (55) Similarly, a succinimide residue has been shown to contribute to the substrate recognition of amylomaltase. (56) Our results demonstrate that nature similarly exploits isoAsp formation to access unusual structures and thereby tailor protein structure and function. This mode of action may be enhanced by the possibility of enzymatic isoAsp formation (57,58) and complements previously studied systems where slow asparagine deamidation and/or isoAsp accumulation regulates biochemical function. (2) Examples include apoptosis, (59,60) cell adhesion, (61) host recognition, (62) as well as the potential for modulation of transcription and translation through molecular aging of histones (63) and ribosomes. (64) The continued discovery of proteins featuring isoAsp or succinimide linkages, as well as the ease in which MurA incorporates and utilizes its isoAsp residue, highlights that such backbone modifications contribute favorably to the structural diversity of proteins. Hence, we speculate that isoAsp-containing hairpins are more common than anticipated and that their stabilizing effects could be harnessed in protein engineering efforts.

Supporting Information

Click to copy section linkSection link copied!

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biochem.0c00502.

  • Additional data and figures including reanalysis of MurA structures, AdoHcy calibration curve, analysis of the reversibility of chemical denaturation, protein characterization (size-exclusion chromatograms, SDS-PAGE and mass spectra), peptide characterization (LC-MS and MS/MS spectra), deamidation analysis (HPLC traces), peptide mapping, CD spectra, ion mobility mass spectra, and Woods plots for HDX experiments (PDF)

Accession Codes

P33038.

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
  • Authors
    • Tianze Zhang - Department of Chemistry, King’s College London, 7 Trinity Street, London SE1 1DB, United Kingdom
    • Kjetil Hansen - Department of Chemistry, King’s College London, 7 Trinity Street, London SE1 1DB, United Kingdom;  Orcidhttp://orcid.org/0000-0002-0085-8440
    • Argyris Politis - Department of Chemistry, King’s College London, 7 Trinity Street, London SE1 1DB, United Kingdom;  Orcidhttp://orcid.org/0000-0002-6658-3224
  • Author Contributions

    M.M.M. conceived the project. T.Z., K.H., A.P., and M.M.M. designed research and analyzed data. T.Z. and K.H. performed experiments with guidance from A.P. and M.M.M. All authors contributed to the writing of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

Click to copy section linkSection link copied!

This work was supported by the Wellcome Trust and the Royal Society (Sir Henry Dale Fellowship 202250/Z/16/Z to M.M.M.), Waters Corporation (Grant to A.P.), China Scholarship Council (Studentship to T.Z.), and the London Interdisciplinary Doctoral Programme, Waters & King’s College London for funding to K.H. The authors thank Dr. Ernst Schönbrunn for providing the Enterobacter cloacae MurA plasmid; Dr. Heather Findlay and Dr. Nicola Harris for advice on CD spectroscopy; Dr. Camille Metier for providing malachite green molybdate reagents; King’s College London Chemistry Department Facility, Dr. Katia Grira, Dr. Steven Howell, and Dr. Bram Snijders for advice with mass spectrometry; and all members of the Müller lab for helpful discussions.

References

Click to copy section linkSection link copied!

This article references 64 other publications.

  1. 1
    Reissner, K. J. and Aswad, D. W. (2003) Deamidation and isoaspartate formation in proteins: unwanted alterations or surreptitious signals?. Cell. Mol. Life Sci. 60, 1281– 1295,  DOI: 10.1007/s00018-003-2287-5
    Google Scholar
    1
    Deamidation and isoaspartate formation in proteins: Unwanted alterations or surreptitious signals?
    Reissner, K. J.; Aswad, D. W.
    Cellular and Molecular Life Sciences (2003), 60 (7), 1281-1295CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
    A review and discussion. The formation of β-linked Asp-Xaa peptide bonds [isoaspartyl (isoAsp) sites] arise in proteins via succinimide-linked deamidation of Asn or dehydration of Asp residues, reactions which represent a major source of spontaneous protein damage under physiol. conditions. Accumulation of atypical isoAsp sites is minimized in vivo by the activity of protein L-isoaspartyl O-methyltransferase (PIMT), which regenerates a normal peptide bond. The loss of PIMT has harmful consequences, esp. in neurons; thus, formation of isoAsp sites and their subsequent correction by PIMT is widely believed to constitute an important pathway of protein damage and repair. Recent evidence is mounting, however, that deamidation and isoAsp formation may, in some instances, constitute a novel mechanism for intentional modification of protein structure. Here, the authors describe the mechanism of Asx rearrangement, summarize evidence that PIMT serves an important repair function, and then focus on emerging evidence that deamidation and isoAsp formation may sometimes have a useful function.
  2. 2
    Robinson, N. E., and Robinson, A. (2004) Molecular clocks: deamidation of asparaginyl and glutaminyl residues in peptides and proteins, Althouse Press.
    Google Scholar
    There is no corresponding record for this reference.
  3. 3
    Eschenburg, S. and Schonbrunn, E. (2000) Comparative X-ray analysis of the un-liganded fosfomycin-target MurA. Proteins: Struct., Funct., Genet. 40, 290– 298,  DOI: 10.1002/(SICI)1097-0134(20000801)40:2<290::AID-PROT90>3.0.CO;2-0
    Google Scholar
    3
    Comparative X-ray analysis of the un-liganded fosfomycin-target MurA
    Eschenburg, Susanne; Schonbrunn, Ernst
    Proteins: Structure, Function, and Genetics (2000), 40 (2), 290-298CODEN: PSFGEY; ISSN:0887-3585. (Wiley-Liss, Inc.)
    MurA, an essential enzyme for the synthesis of the bacterial cell wall, follows an induced-fit mechanism. Upon substrate binding, the active site forms in the interdomain cleft, involving movements of the two domains of the protein and a reorientation of the loop Pro112-Pro121. We compare two structures of un-liganded MurA from Enterobacter cloacae: a new orthorhombic form, solved to 1.80 Å resoln., and a monoclinic form, redetd. to 1.55 Å resoln. In the monoclinic form, the loop Pro112-Pro121 stretches into solvent, while in the new form it adopts a wound conformation, thereby reducing solvent accessibility of the crit. residue Cys115. In the interdomain cleft a network of 27 common water mols. has been identified, which partially shields neg. charges in the cleft and stabilizes the orientation of catalytically crucial residues. This could support substrate binding and ease domain movements. Near the hinge region an isoaspartyl residue has been recognized, which is the product of post-translational modification of the genetically encoded Asn67-Gly68. The homogeneous population with L-isoaspartate in both structures suggests that the modification in Enterobacter cloacae MurA is not a mere aging defect but rather the result of a specific in vivo process.
  4. 4
    Robinson, N. E. and Robinson, A. B. (2001) Molecular clocks. Proc. Natl. Acad. Sci. U. S. A. 98, 944– 949,  DOI: 10.1073/pnas.98.3.944
    Google Scholar
    4
    Molecular clocks
    Robinson, Noah E.; Robinson, Arthur B.
    Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (3), 944-949CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    A convenient and precise mass spectrometric method for measurement of the deamidation rates of glutaminyl and asparaginyl residues in peptides and proteins has been developed; the rates of deamidation of 306 asparaginyl sequences in model peptides at pH 7.4, 37.0°, 0.15 M Tris·HCl buffer have been detd.; a library of 913 amide-contg. peptides for use by other investigators in similar studies has been established; and, by means of simultaneous deamidation rate measurements of rabbit muscle aldolase and appropriate model peptides in the same solns., the use of this method for quant. measurement of the relative effects of primary, secondary, tertiary, and quaternary protein structure on deamidation rates has been demonstrated. The measured rates are discussed with respect to the hypothesis that glutaminyl and asparaginyl residues serve, through deamidation, as mol. timers of biol. events.
  5. 5
    Xie, M. and Schowen, R. L. (1999) Secondary Structure and Protein Deamidation. J. Pharm. Sci. 88, 8– 13,  DOI: 10.1021/js9802493
    Google Scholar
    5
    Secondary Structure and Protein Deamidation
    Xie, Minli; Schowen, Richard L.
    Journal of Pharmaceutical Sciences (1999), 88 (1), 8-13CODEN: JPMSAE; ISSN:0022-3549. (American Chemical Society)
    The deamidation reactions of asparagine residues in α-helical and β-turn secondary structural environments of peptides and proteins are reviewed with 31 refs. Both kinds of secondary structure tend to stabilize asparagine residues against deamidation, although the effects are not large. The effect of β-sheet structures on asparagine stability is unclear, although simple considerations suggest a stabilization in this environment also.
  6. 6
    Shimizu, T., Matsuoka, Y., and Shirasawa, T. (2005) Biological Significance of Isoaspartate and Its Repair System. Biol. Pharm. Bull. 28, 1590– 1596,  DOI: 10.1248/bpb.28.1590
    Google Scholar
    6
    Biological significance of isoaspartate and its repair system
    Shimizu, Takahiko; Matsuoka, Yasuji; Shirasawa, Takuji
    Biological & Pharmaceutical Bulletin (2005), 28 (9), 1590-1596CODEN: BPBLEO; ISSN:0918-6158. (Pharmaceutical Society of Japan)
    A review. The isomerization of L-aspartate and the deamidation of L-asparagine in proteins or peptides dominantly give rise to L-isoaspartate by a nonenzymic reaction via succinimide as a intermediate under physiol. conditions. Isoaspartates have been identified in a variety of cellular proteins in vivo as well as pathol. deposited proteins in neurodegenerative brain tissue. Here, the authors describe the enhanced formation of isoaspartate in amyloid-β (Aβ) peptides in Alzheimer's disease (AD). Specific antibodies recognizing isoaspartate in Aβ have revealed that isomerized Aβ peptides are deposited in senile plaques as well as amyloid-bearing vessels. Moreover, it has been obsd. that Aβ peptides, isomerized at positions 7 or 23, are differentially deposited in senile plaques and vascular amyloids in AD brains. In vitro expts. have shown that the modification at position 23 greatly enhances the aggregation of Aβ. Furthermore, systematic proline substitution analyses has revealed that the β-turn structure at positions 22 and 23 of Aβ42 plays a crucial role in the aggregation and neurotoxicity of Aβ peptides. It is suggested that spontaneous isomerization at position 23 induces the conformational change to form a β-turn at position 23, which plays a pathogenic role in the deposition of Aβ peptides in sporadic AD. Protein L-isoaspartyl methyltransferase (PIMT) is a putative protein repair enzyme, which converts L-isoaspartyl residues in damaged proteins to normal L-aspartyl residues. PIMT-deficient mice have shown neurodegenerative changes concomitant with the accumulation of L-isoaspartate in the brain. The authors discuss the pathol. implications of the formation of isoaspartate in damaged proteins during neurodegeneration in model mice and AD.
  7. 7
    Fukuda, H., Shimizu, T., Nakajima, M., Mori, H., and Shirasawa, T. (1999) Synthesis, aggregation, and neurotoxicity of the alzheimer’s Aß1–42 amyloid peptide and its isoaspartyl isomers. Bioorg. Med. Chem. Lett. 9, 953– 956,  DOI: 10.1016/S0960-894X(99)00121-3
    Google Scholar
    7
    Synthesis, aggregation, and neurotoxicity of the Alzheimer's Aβ1-42 amyloid peptide and its isoaspartyl isomers
    Fukuda, Hiroyuki; Shimizu, Takahiko; Nakajima, Mitsunari; Mori, Hiroshi; Shirasawa, Takuji
    Bioorganic & Medicinal Chemistry Letters (1999), 9 (7), 953-956CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)
    Amyloid Aβ1-42 peptide (Aβ1-42) and its isomers with an isoaspartyl residue at position 7 or 23 [Aβ1-42(isoAsp7) and Aβ1-42(isoAsp23)] were synthesized in high purity by the Fmoc-solid phase technique, followed by HPLC on a silica-based reversed-phase column under the basic conditions. Aβ1-42(isoAsp23) aggregated more strongly than native Aβ1-42 and showed significant neurotoxicity, while the aggregation ability and neurotoxicity of Aβ1-42(isoAsp7) was weak. This suggests that the isomerization of the aspartyl residues plays an important role in fibril formation in Alzheimer's disease.
  8. 8
    Shimizu, T., Watanabe, A., Ogawara, M., Mori, H., and Shirasawa, T. (2000) Isoaspartate formation and neurodegeneration in Alzheimer’s disease. Arch. Biochem. Biophys. 381, 225– 234,  DOI: 10.1006/abbi.2000.1955
    Google Scholar
    8
    Isoaspartate formation and neurodegeneration in Alzheimer's disease
    Shimizu, Takahiko; Watanabe, Atsushi; Ogawara, Midori; Mori, Hiroshi; Shirasawa, Takuji
    Archives of Biochemistry and Biophysics (2000), 381 (2), 225-234CODEN: ABBIA4; ISSN:0003-9861. (Academic Press)
    A review with 99 refs. We reviewed here that protein isomerization is enhanced in amyloid-β peptides (Aβ) and paired helical filaments (PHFs) purified from Alzheimer's disease (AD) brains. Biochem. analyses revealed that Aβ purified from senile plaques and vascular amyloid are isomerized at Asp-1 and Asp-7. A specific antibody recognizing isoAsp-23 of Aβ further suggested the isomerization of Aβ at Asp-23 in vascular amyloid as well as in the core of senile plaques. Biochem. analyses of purified PHFs also revealed that heterogeneous mol. wt. tau contains L-isoaspartate at Asp-193, Asn-381, and Asp-387, indicating a modification, other than phosphorylation, that differentiates between normal tau and PHF tau. Since protein isomerization as L-isoaspartate causes structural changes and functional inactivation, or enhances the aggregation process, this modification is proposed as one of the progression factors in AD. Protein L-isoaspartyl methyltransferase (PIMT) is suggested to play a role in the repair of isomerized proteins contg. L-isoaspartate. We show here that PIMT is upregulated in neurodegenerative neurons and colocalizes in neurofibrillary tangles (NFTs) in AD. Taken together with the enhanced protein isomerization in AD brains, it is implicated that the upregulated PIMT may assoc. with increased protein isomerization in AD. We also reviewed studies on PIMT-deficient mice that confirmed that PIMT plays a physiol. role in the repair of isomerized proteins contg. L-isoaspartate. The knockout study also suggested that the brain of PIMT-deficient mice manifested neurodegenerative changes concomitant with accumulation of L-isoaspartate. We discuss the pathol. implications of protein isomerization in the neurodegeneration found in model mice and AD. (c) 2000 Academic Press.
  9. 9
    Warmack, R. A., Boyer, D. R., Zee, C.-T., Richards, L. S., Sawaya, M. R., Cascio, D., Gonen, T., Eisenberg, D. S., and Clarke, S. G. (2019) Structure of amyloid-β (20–34) with Alzheimer’s-associated isomerization at Asp23 reveals a distinct protofilament interface. Nat. Commun. 10, 3357,  DOI: 10.1038/s41467-019-11183-z
    Google Scholar
    9
    Structure of amyloid-β (20-34) with Alzheimer's-associated isomerization at Asp23 reveals a distinct protofilament interface
    Warmack Rebeccah A; Boyer David R; Zee Chih-Te; Richards Logan S; Sawaya Michael R; Cascio Duilio; Eisenberg David S; Clarke Steven G; Sawaya Michael R; Cascio Duilio; Gonen Tamir; Eisenberg David S; Clarke Steven G; Sawaya Michael R; Cascio Duilio; Eisenberg David S; Gonen Tamir; Eisenberg David S; Gonen Tamir; Eisenberg David S; Gonen Tamir
    Nature communications (2019), 10 (1), 3357 ISSN:.
    Amyloid-β (Aβ) harbors numerous posttranslational modifications (PTMs) that may affect Alzheimer's disease (AD) pathogenesis. Here we present the 1.1 ÅA resolution MicroED structure of an Aβ 20-34 fibril with and without the disease-associated PTM, L-isoaspartate, at position 23 (L-isoAsp23). Both wild-type and L-isoAsp23 protofilaments adopt β-helix-like folds with tightly packed cores, resembling the cores of full-length fibrillar Aβ structures, and both self-associate through two distinct interfaces. One of these is a unique Aβ interface strengthened by the isoaspartyl modification. Powder diffraction patterns suggest a similar structure may be adopted by protofilaments of an analogous segment containing the heritable Iowa mutation, Asp23Asn. Consistent with its early onset phenotype in patients, Asp23Asn accelerates aggregation of Aβ 20-34, as does the L-isoAsp23 modification. These structures suggest that the enhanced amyloidogenicity of the modified Aβ segments may also reduce the concentration required to achieve nucleation and therefore help spur the pathogenesis of AD.
  10. 10
    Lampi, K. J., Wilmarth, P. A., Murray, M. R., and David, L. L. (2014) Lens β-crystallins: the role of deamidation and related modifications in aging and cataract. Prog. Biophys. Mol. Biol. 115, 21– 31,  DOI: 10.1016/j.pbiomolbio.2014.02.004
    Google Scholar
    10
    Lens β-crystallins: The role of deamidation and related modifications in aging and cataract
    Lampi, Kirsten J.; Wilmarth, Phillip A.; Murray, Matthew R.; David, Larry L.
    Progress in Biophysics & Molecular Biology (2014), 115 (1), 21-31CODEN: PBIMAC; ISSN:0079-6107. (Elsevier Ltd.)
    A review. Crystallins are the major proteins in the lens of the eye and function to maintain transparency of the lens. Of the human crystallins, α, β, and γ, the β-crystallins remain the most elusive in their structural significance due to their greater no. of subunits and possible oligomer formations. The β-crystallins are also heavily modified during aging. This review focuses on the functional significance of deamidation and the related modifications of racemization and isomerization, the major modifications in β-crystallins of the aged human lens. Elucidating the role of these modifications in cataract formation has been slow, because they are anal. among the most difficult post-translational modifications to study. Recent results suggest that many amides deamidate to similar extent in normal aged and cataractous lenses, while others may undergo greater deamidation in cataract. Mimicking deamidation at crit. structural regions induces structural changes that disrupt the stability of the β-crystallins and lead to their aggregation in vitro. Deamidations at the surface disrupt interactions with other crystallins. Addnl., the α-crystallin chaperone is unable to completely prevent deamidated β-crystallins from insolubilization. Therefore, deamidation of β-crystallins may enhance their pptn. and light scattering in vivo contributing to cataract formation.Future expts. are needed to quantify differences in deamidation rates at all Asn and Gln residues within crystallins from aged and cataractous lenses, as well as racemization and isomerization which potentially perturb protein structure greater than deamidation alone. Quant. data is greatly needed to investigate the importance of these major age-related modifications in cataract formation.
  11. 11
    McFadden, P. N. and Clarke, S. (1987) Conversion of isoaspartyl peptides to normal peptides: implications for the cellular repair of damaged proteins. Proc. Natl. Acad. Sci. U. S. A. 84, 2595– 2599,  DOI: 10.1073/pnas.84.9.2595
    Google Scholar
    11
    Conversion of isoaspartyl peptides to normal peptides: implications for the cellular repair of damaged proteins
    McFadden, Philip N.; Clarke, Steven
    Proceedings of the National Academy of Sciences of the United States of America (1987), 84 (9), 2595-9CODEN: PNASA6; ISSN:0027-8424.
    The hypothesis that cellular protein carboxymethylation reactions recognize altered aspartyl residues as part of a protein repair pathway has been tested in an in vitro system using tetragastrin (Trp-Met-Asp-Phe-NH2) as a model sequence. The L-isoaspartyl form of tetragastrin, where the phenylalanine residue is linked to the side-chain CO2H group of the aspartate residue ([iso-Asp3]tetragastrin), is a substrate for the erythrocyte protein carboxyl methyltransferases, whereas the normal form is not. The enzymically produced α-Me ester of [iso-Asp3]tetragastrin, [iso-Asp(OMe)3]tetragastrin, is unstable at pH 7.4 and 37° and spontaneously demethylates with a half-time of 41 min to an intermediate L-succinimide form ([Asu3]tetragastrin) that, in turn, spontaneously hydrolyzes with a half-time of 116 min to give a mixt. of normal tetragastrin (20%) and [iso-Asp3]tetragastrin (80%). This sequence of enzymic and nonenzymic reactions can be coupled in a single reaction mixt.; the [iso-Asp3]tetragastrin that is produced upon succinimide hydrolysis can reenter the reaction sequence by enzymic methylation, and the net result of the process is the conversion of the isomerized peptide to the normal peptide. The efficiency of this repair reaction is limited by a side reaction of racemization at the α-C of the succinimide (half-time = 580 min). In a 24-h time period, normal L-aspartyl-contg. tetragastrin is obtained in ∼50% yield from the coupled reaction mixt.; other products include [D-iso-Asp3]tetragastrin and [D-Asp3]tetragastrin. The versatile chem. of succinimide peptides suggests that methylated L-isoaspartyl sites (and possibly methylated D-aspartyl sites) in cellular polypeptides can eventually yield repaired normal L-aspartyl sites through succinimide intermediates.
  12. 12
    Johnson, B. A., Murray, E. D., Clarke, S., Glass, D. B., and Aswad, D. W. (1987) Protein carboxyl methyltransferase facilitates conversion of atypical L-isoaspartyl peptides to normal L-aspartyl peptides. J. Biol. Chem. 262, 5622– 5629
    Google Scholar
    12
    Protein carboxyl methyltransferase facilitates conversion of atypical L-isoaspartyl peptides to normal L-aspartyl peptides
    Johnson, Brett A.; Murray, E. David, Jr.; Clarke, Steven; Glass, David B.; Aswad, Dana W.
    Journal of Biological Chemistry (1987), 262 (12), 5622-9CODEN: JBCHA3; ISSN:0021-9258.
    Prolonged incubation of L-isoaspartate (L-iso-Asp)-contg. forms of lactate dehydrogenase (231-242), sperm activating peptide, and ACTH (22-27) at 37°, pH 7.4, with S-adenosyl-L-methionine and protein carboxyl methyltransferase (I) from bovine brain leads to extensive conversion of the atypical isopeptide bond to a normal peptide bond. For the lactate dehydrogenase-related peptide, conversion was 80% complete after 24 h. For the other 2 peptides, conversion reached a level of ∼65% after 48 h. The mechanism of conversion involved (1) rapid enzymic methylation of the α-CO2H of the L-iso-Asp residue; (2) nonenzymic demethylation resulting in formation of an L-aspartyl cyclic imide; and (3) a slow, nonenzymic hydrolysis of the cyclic imide to form a mixt. of 15-25% normal L-Asp (aspartate) peptide and 75-85% L-iso-Asp peptide. The regenerated L-iso-Asp peptide was remethylated and the cycle was repeated. The extent of conversion was limited by a competing side reaction wherein the L-imide slowly racemizes, leading to the formation of mainly D-iso-Asp peptide, which was not a substrate for I. The ability of I to initiate conversion of L-iso-Asp residues to normal L-Asp suggests a possible role for this enzyme in facilitating the repair or degrdn. of deamidated proteins in vivo.
  13. 13
    Lowenson, J. D., Kim, E., Young, S. G., and Clarke, S. (2001) Limited Accumulation of Damaged Proteins in L-Isoaspartyl (D-Aspartyl) O-Methyltransferase-deficient Mice. J. Biol. Chem. 276, 20695– 20702,  DOI: 10.1074/jbc.M100987200
    Google Scholar
    13
    Limited accumulation of damaged proteins in L-isoaspartyl (D-aspartyl) O-methyltransferase-deficient mice
    Lowenson, Jonathan D.; Kim, Edward; Young, Stephen G.; Clarke, Steven
    Journal of Biological Chemistry (2001), 276 (23), 20695-20702CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    L-Isoaspartyl (D-aspartyl) O-methyltransferase (PCMT1) can initiate the conversion of damaged aspartyl and asparaginyl residues to normal L-aspartyl residues. Mice lacking this enzyme (Pcmt1 -/- mice) have elevated levels of damaged residues and die at a mean age of 42 days from massive tonic-clonic seizures. To extend the lives of the knockout mice so that the long term effects of damaged residue accumulation could be investigated, we produced transgenic mice with a mouse Pcmt1 cDNA under the control of a neuron-specific promoter. Pcmt1 transgenic mice that were homozygous for the endogenous Pcmt1 knockout mutation ("transgenic Pcmt1 -/- mice") had brain PCMT1 activity levels that were 6.5-13% those of wild-type mice but had little or no activity in other tissues. The transgenic Pcmt1 -/- mice lived, on av., 5-fold longer than nontransgenic Pcmt1 -/- mice and accumulated only half as many damaged aspartyl residues in their brain proteins. The concn. of damaged residues in heart, testis, and brain proteins in transgenic Pcmt1 -/- mice initially increased with age but unexpectedly reached a plateau by 100 days of age. Urine from Pcmt1 -/- mice contained increased amts. of peptides with damaged aspartyl residues, apparently enough to account for proteins that were not repaired intracellularly. In the absence of PCMT1, proteolysis may limit the intracellular accumulation of damaged proteins but less efficiently than in wild-type mice having PCMT1-mediated repair.
  14. 14
    Yamamoto, A., Takagi, H., Kitamura, D., Tatsuoka, H., Nakano, H., Kawano, H., Kuroyanagi, H., Yahagi, Y.-I., Kobayashi, S.-I., Koizumi, K.-I., Sakai, T., Saito, K.-I., Chiba, T., Kawamura, K., Suzuki, K., Watanabe, T., Mori, H., and Shirasawa, T. (1998) Deficiency in Protein Isoaspartyl Methyltransferase Results in a Fatal Progressive Epilepsy. J. Neurosci. 18, 2063– 2074,  DOI: 10.1523/JNEUROSCI.18-06-02063.1998
    Google Scholar
    14
    Deficiency in protein L-isoaspartyl methyltransferase results in a fatal progressive epilepsy
    Yamamoto, Akihiro; Takagi, Hideyuki; Kitamura, Daisuke; Tatsuoka, Hozumi; Nakano, Hirotake; Kawano, Hitoshi; Kuroyanagi, Hidehito; Yahagi, Yu-Ichi; Kobayashi, Shin-Ichiro; Koizumi, Ken-Ichi; Sakai, Tsuyoshi; Saito, Ken-Ichi; Chiba, Tanemichi; Kawamura, Koki; Suzuki, Katsushi; Watanabe, Takeshi; Mori, Hiroshi; Shirasawa, Takuji
    Journal of Neuroscience (1998), 18 (6), 2063-2074CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
    Protein L-isoaspartyl methyltransferase (PIMT) is suggested to play a role in the repair of aged protein spontaneously incorporated with isoaspartyl residues. The authors generated PIMT-deficient mice by targeted disruption of the PIMT gene to elucidate the biol. role of the gene in vivo. PIMT-deficient mice died from progressive epileptic seizures with grand mal and myoclonus between 4 and 12 wk of age. An anticonvulsive drug, dipropylacetic acid (DPA), improved their survival but failed to cure the fatal outcome. L-Isoaspartate, the putative substrate for PIMT, was increased ninefold in the brains of PIMT-deficient mice. The brains of PIMT-deficient mice started to enlarge after 4 wk of age when the apical dendrites of pyramidal neurons in cerebral cortices showed aberrant arborizations with disorganized microtubules. The authors conclude that methylation of modified proteins with isoaspartyl residues is essential for the maintenance of a mature CNS and that a deficiency in PIMT results in fatal progressive epilepsy in mice.
  15. 15
    Heijenoort, J. V. (2001) Recent advances in the formation of the bacterial peptidoglycan monomer unit. Nat. Prod. Rep. 18, 503– 519,  DOI: 10.1039/a804532a
    Google Scholar
    There is no corresponding record for this reference.
  16. 16
    Seebach, D., Beck, A. K., and Bierbaum, D. J. (2004) The World of β- and γ-Peptides Comprised of Homologated Proteinogenic Amino Acids and Other Components. Chem. Biodiversity 1, 1111– 1239,  DOI: 10.1002/cbdv.200490087
    Google Scholar
    16
    The world of β- and γ-peptides comprised of homologated proteinogenic amino acids and other components
    Seebach, Dieter; Beck, Albert K.; Bierbaum, Daniel J.
    Chemistry & Biodiversity (2004), 1 (8), 1111-1239CODEN: CBHIAM; ISSN:1612-1872. (Verlag Helvetica Chimica Acta AG)
    A review. The origins of our nearly ten-year research program of chem. and biol. investigations into peptides based on homologated proteinogenic amino acids are described. The road from the biopolymer poly[ethyl (R)-3-hydroxybutanoate] to the β-peptides was primarily a step from org. synthesis methodol. (the prepn. of enantiomerically pure compds. (EPCs)) to supramol. chem. (higher-order structures maintained through non-covalent interactions). The performing of biochem. and biol. tests on the β-and γ-peptides, which differ from natural peptides/proteins by a single or two addnl. CH2 groups per amino acid, then led into bioorg. chem. and medicinal chem. The individual chapters of this review article begin with descriptions of work on β-amino acids, β-peptides, and polymers (Nylon-3) that dates back to the 1960s, even to the times of Emil Fischer, but did not yield insights into structures or biol. properties. The numerous, often highly physiol. active, or even toxic, natural products contg. β- and γ-amino acid moieties are then presented. Chapters on the prepn. of homologated amino acids with proteinogenic side chains, their coupling to provide the corresponding peptides, both in soln. (including thioligation) and on the solid phase, their isolation by preparative HPLC, and their characterization by mass spectrometry (HR-MS and MS sequencing) follow. After that, their structures, predominantly detd. by NMR spectroscopy in methanolic soln., are described: helixes, pleated sheets, and turns, together with stack-, crankshaft-, paddlewheel-, and staircase-like patterns. The presence of the addnl. C-C bonds in the backbones of the new peptides did not give rise to a chaotic increase in their secondary structures as many protein specialists might have expected: while there are indeed more structure types than are obsd. in the α-peptide realm - three different helixes (10/12-, 12-, and 14-helix) if we include oligomers of trans-2-aminocyclopentanecarboxylic acid, for example - the structures are already observable with chains made up of only four components, and, having now undergone a learning process, we are able to construct them by design. The structures of the shorter β-peptides can also be reliably detd. by mol.-dynamics calcns. (in soln.; GROMOS program package). Unlike in the case of the natural helixes, these compds.' folding into secondary structures is not cooperative. In β- and γ-peptides, it is possible to introduce heteroatom substituents (such as halogen or OH) onto the backbones or to incorporate heteroatoms (NH, O) directly into the chain, and, thanks to this, it has been possible to study effects unobservable in the world of the α-peptides. Tests with proteolytic enzymes of all types (from mammals, microorganisms, yeasts) and in vivo examn. (mice, rats, insects, plants) showed β- and γ-peptides to be completely stable towards proteolysis and, as demonstrated for two β-peptides, extraordinarily stable towards metab., even when bearing functionalized side chains (such as those of Thr, Tyr, Trp, Lys, or Arg). The β-peptides so far examd. also normally display no or only very weak cytotoxic, antiproliferative, antimicrobial, hemolytic, immunogenic, or inflammatory properties either in cell cultures or in vivo. Even biol. degrdn. by microbial colonies of the types found in sewage-treatment plants or in soil is very slow. Besides these less sharply definable interactions, it has also been possible to construct β- and γ-peptide agonists of naturally occurring peptide hormones, MHC-binding β-peptides, or amphipathic β-peptide inhibitors of membrane-bound proteins in a controlled fashion. Examples include somatostatin mimics and the suppression of cholesterol transport through the intestinal brush-border membrane (by the SR-BI-protein). The results so far obtained from investigations into peptides made up of homologues of the proteinogenic amino acids also represent a contribution to deepening of our knowledge of the natural peptides/proteins, while potential for biomedicinal application of this new class of substances has also been suggested.
  17. 17
    Gellman, S. H. (1998) Foldamers: A manifesto. Acc. Chem. Res. 31, 173– 180,  DOI: 10.1021/ar960298r
    Google Scholar
    17
    Foldamers: A Manifesto
    Gellman, Samuel H.
    Accounts of Chemical Research (1998), 31 (4), 173-180CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review with 79 refs. on the indentification of new polymeric systems, termed foldamers, with strong tendencies to adopt specific compact conformations, including the development of predictively useful understanding of the relationship between the repetitive features of the monomer structure and conformational properties at the polymer level. Topics include background, case studies of β-peptides, DNA and RNA analogs, and other foldamers.
  18. 18
    Minasov, G., Halavaty, A., Filippova, E. V., Shuvalova, L., Dubrovska, I., Winsor, J., Papazisi, L., and Anderson, W. F. (2011) 2.6 Angstrom Crystal Structure of UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1 (MurA1) from Bacillus anthracis. Worldwide Protein Data Bank 3SG1,  DOI: 10.2210/pdb3SG1/pdb
    Google Scholar
    There is no corresponding record for this reference.
  19. 19
    Yoon, H. J., Lee, S. J., Mikami, B., Park, H. J., Yoo, J., and Suh, S. W. (2008) Crystal structure of UDP-N-acetylglucosamine enolpyruvyl transferase from Haemophilus influenzae in complex with UDP-N-acetylglucosamine and fosfomycin. Proteins: Struct., Funct., Genet. 71, 1032– 1037,  DOI: 10.1002/prot.21959
    Google Scholar
    19
    Crystal structure of UDP-N-acetylglucosamine enolpyruvyl transferase from Haemophilus influenzae in complex with UDP-N-acetylglucosamine and fosfomycin
    Yoon, Hye-Jin; Lee, Sang Jae; Mikami, Bunzo; Park, Hyun-Ju; Yoo, Jakyung; Suh, Se Won
    Proteins: Structure, Function, and Bioinformatics (2008), 71 (2), 1032-1037CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
    Peptidoglycan (also known as murein) serves a structural role in the bacterial cell wall. The bacterial enzyme UDP-N-acetylglucosamine enolpyruyl transferase (MurA; also called MurZ; E.C. 2.5.1.7) catalyzes the first committed step of peptidoglycan biosynthesis, that is transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine to form enolpyruyl-UDP-N-acetylglucosamine. We have initiated structure detn. of the 424-residue MurA from H. influenzae. Here we report two crystal structures of H. influenzae MurA: a binary complex with the substrate UDP-N-acetylglucosamine at 2.2 Å resoln. and a ternary complex with UDP-N-acetylglucosamine and fosfomycin at 2.3 Å resoln. Our binary complex structure is the first structure of MurA bound with the substrate UDP- N-acetylglucosamine and thus our study complements the previous structures. The binary and ternary complex structures of H. influenzae MurA are highly similar to each other, both displaying a half-open conformation for the active site loop. This is in sharp contrast with the closed conformation of the corresponding loop in the E. coli MurA ternary complex with UDP-N-acetylglucosamine and fosfomycin. Our results will be useful in structure-based design of specific inhibitors of MurA for antibacterial discovery.
  20. 20
    Minasov, G., Shuvalova, L., Dubrovska, I., Kiryukhina, O., Grimshaw, S., Kwon, K., and Anderson, W. F. (2017) 2.0 Angstrom Resolution Crystal Structure of UDP-N-acetylglucosamine 1-carboxyvinyltransferase from Streptococcus pneumoniae in Complex with Uridine-diphosphate-2(n-acetylglucosaminyl) butyric acid, (2R)-2-(phosphonooxy)propanoic acid and Magnesium. Worldwide Protein Data Bank 5WI5,  DOI: 10.2210/pdb5WI5/pdb
    Google Scholar
    There is no corresponding record for this reference.
  21. 21
    Schurter, B. T. and Aswad, D. W. (2000) Analysis of isoaspartate in peptides and proteins without the use of radioisotopes. Anal. Biochem. 282, 227– 231,  DOI: 10.1006/abio.2000.4601
    Google Scholar
    21
    Analysis of Isoaspartate in Peptides and Proteins without the Use of Radioisotopes
    Schurter, Brandon T.; Aswad, Dana W.
    Analytical Biochemistry (2000), 282 (2), 227-231CODEN: ANBCA2; ISSN:0003-2697. (Academic Press)
    A rapid and sensitive HPLC-based method for quantitating isoaspartate levels in peptides and proteins is described. The analyte is incubated for 40 min with S-adenosyl-l-methionine and the com. available enzyme protein L-isoaspartyl methyltransferase. Methylation of isoaspartyl sites results in stoichiometric prodn. of S-adenosyl-L-homocysteine that is sepd. from the other components of the reaction by reversed-phase HPLC and quantitated online by absorbance at 260 nm. This method can accurately detect 5 pmol or less of isoaspartate and works with tryptic digests as well as intact proteins. Using a com. available isoaspartyl peptide, the relationship between isoaspartate levels and S-adenosyl-l-homocysteine prodn. was found to be linear and stoichiometric over a range of 5-250 pmol. Compared to methods that measure [3H]methanol prodn. after methylation with S-adenosyl-l-[methyl-3H]methionine, the HPLC method is safer, faster, less expensive, and equally sensitive. (c) 2000 Academic Press.
  22. 22
    Masson, G. R., Burke, J. E., Ahn, N. G., Anand, G. S., Borchers, C., Brier, S., Bou-Assaf, G. M., Engen, J. R., Englander, S. W., Faber, J., Garlish, R., Griffin, P. R., Gross, M. L., Guttman, M., Hamuro, Y., Heck, A. J. R., Houde, D., Iacob, R. E., Jorgensen, T. J. D., Kaltashov, I. A., Klinman, J. P., Konermann, L., Man, P., Mayne, L., Pascal, B. D., Reichmann, D., Skehel, M., Snijder, J., Strutzenberg, T. S., Underbakke, E. S., Wagner, C., Wales, T. E., Walters, B. T., Weis, D. D., Wilson, D. J., Wintrode, P. L., Zhang, Z., Zheng, J., Schriemer, D. C., and Rand, K. D. (2019) Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments. Nat. Methods 16, 595– 602,  DOI: 10.1038/s41592-019-0459-y
    Google Scholar
    22
    Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments
    Masson, Glenn R.; Burke, John E.; Ahn, Natalie G.; Anand, Ganesh S.; Borchers, Christoph; Brier, Sebastien; Bou-Assaf, George M.; Engen, John R.; Englander, S. Walter; Faber, Johan; Garlish, Rachel; Griffin, Patrick R.; Gross, Michael L.; Guttman, Miklos; Hamuro, Yoshitomo; Heck, Albert J. R.; Houde, Damian; Iacob, Roxana E.; Joergensen, Thomas J. D.; Kaltashov, Igor A.; Klinman, Judith P.; Konermann, Lars; Man, Petr; Mayne, Leland; Pascal, Bruce D.; Reichmann, Dana; Skehel, Mark; Snijder, Joost; Strutzenberg, Timothy S.; Underbakke, Eric S.; Wagner, Cornelia; Wales, Thomas E.; Walters, Benjamin T.; Weis, David D.; Wilson, Derek J.; Wintrode, Patrick L.; Zhang, Zhongqi; Zheng, Jie; Schriemer, David C.; Rand, Kasper D.
    Nature Methods (2019), 16 (7), 595-602CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
    Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophys. technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, anal., presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further addns. and refinements as the field advances.
  23. 23
    Lau, A. M. C., Ahdash, Z., Martens, C., and Politis, A. (2019) Deuteros: software for rapid analysis and visualization of data from differential hydrogen deuterium exchange-mass spectrometry. Bioinformatics 35, 3171– 3173,  DOI: 10.1093/bioinformatics/btz022
    Google Scholar
    23
    Deuteros: software for rapid analysis and visualization of data from differential hydrogen deuterium exchange-mass spectrometry
    Lau, Andy M. C.; Ahdash, Zainab; Martens, Chloe; Politis, Argyris
    Bioinformatics (2019), 35 (17), 3171-3173CODEN: BOINFP; ISSN:1367-4811. (Oxford University Press)
    Hydrogen deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful technique for interrogating the conformational dynamics of proteins and their complexes. Currently, anal. of HDX-MS data remains a laborious procedure, mainly due to the lack of streamlined software to process the large datasets. We present Deuteros which is a standalone software designed to be coupled with Waters DynamX HDX data anal. software, allowing the rapid anal. and visualization of data from differential HDX-MS.
  24. 24
    Brown, E. D., Vivas, E. I., Walsh, C. T., and Kolter, R. (1995) MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli. J. Bacteriol. 177, 4194– 4197,  DOI: 10.1128/JB.177.14.4194-4197.1995
    Google Scholar
    24
    MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli
    Brown, Eric D.; Vivas, Eugenio I.; Walsh, Christopher T.; Kolter, Roberto
    Journal of Bacteriology (1995), 177 (14), 4194-7CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    The E. coli gene murZ was recently shown to encode UDP-N-acetylglucosamine enolpyruvyl transferase, which catalyzes the 1st committed step of peptidoglycan biosynthesis. The map position of murZ (69.3 min) differed from that detd. for murA (90 min), a gene which had been previously proposed to encode the same activity. Here, the construction of a chromosomal deletion of murZ and a plasmid contg. murZ under arabinose control is described. Growth of cells contg. the murZ deletion was dependent on the expression of murZ from the plasmid. MurZ apparently is an essential gene and encodes the sole UDP-N-acetylglucosamine enolpyruvyl transferase of E. coli. To simplify the nomenclature, it is recommended that murA be used to designate the gene at 69.3 min that encodes this activity and that the designation murZ be abandoned.
  25. 25
    Johnson, B. A. and Aswad, D. W. (1991) Optimal conditions for the use of protein L-isoaspartyl methyltransferase in assessing the isoaspartate content of peptides and proteins. Anal. Biochem. 192, 384– 391,  DOI: 10.1016/0003-2697(91)90553-6
    Google Scholar
    25
    Optimal conditions for the use of protein L-isoaspartyl methyltransferase in assessing the isoaspartate content of peptides and proteins
    Johnson, Brett A.; Asward, Dana W.
    Analytical Biochemistry (1991), 192 (2), 384-91CODEN: ANBCA2; ISSN:0003-2697.
    Protein L-isoaspartyl methyltransferase provides a basis for enzymic measurement of atypical, isoaspartyl linkages which make a major contribution to protein microheterogeneity. The low Vmax of the methyltransferase reaction and the instability of the Me ester can hinder accurate detns., and different labs. using different conditions have achieved discrepant values for the isoaspartate content of the same proteins. To investigate the effects of these conditions, and to optimize the assay, isoaspartyl δ sleep-inducing peptide was methylated under a variety of conditions. One μM methyltransferase was required to obtain stoichiometric modification of 2 μM peptide in 40-min reactions at pH 6.2 and 30°. A computer model utilizing kinetic consts. obtained from studies on initial rates of methylation predicted the same requirement for enzyme concn. Carrier protein was necessary for optimal methyltransferase activity at enzyme concns. below 0.4 μM. Stoichiometric methylation required concns. of S-adenosylmethionine to be in substantial excess over those of peptide; 50 μM S-adenosylmethionine is the min. needed for complete modification of 10 μM peptide. Spontaneous demethylation was significant under all conditions tested, so that the Me ester itself never reached a ratio of 1 mol/mol of total peptide. These results demonstrate that the most accurate measurements of isoaspartate are obtained when reactions are carried out at low peptide concns., high S-adenosylmethionine concns., and high enzyme concns. Moreover, quantitation will be significantly better when based on measurements of both methanol and Me esters rather than on measurements of intact Me esters alone.
  26. 26
    Griffith, S. C., Sawaya, M. R., Boutz, D. R., Thapar, N., Katz, J. E., Clarke, S., and Yeates, T. O. (2001) Crystal structure of a protein repair methyltransferase from Pyrococcus furiosus with its L-isoaspartyl peptide substrate. J. Mol. Biol. 313, 1103– 1116,  DOI: 10.1006/jmbi.2001.5095
    Google Scholar
    26
    Crystal Structure of a Protein Repair Methyltransferase from Pyrococcus furiosus with its L-Isoaspartyl Peptide Substrate
    Griffith, Scott C.; Sawaya, Michael R.; Boutz, Daniel R.; Thapar, Nitika; Katz, Jonathan E.; Clarke, Steven; Yeates, Todd O.
    Journal of Molecular Biology (2001), 313 (5), 1103-1116CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
    Protein L-isoaspartyl (D-aspartyl) methyltransferases (E.C. 2.1.1.77) are found in almost all organisms. These enzymes catalyze the S-adenosylmethionine (AdoMet)-dependent methylation of isomerized and racemized aspartyl residues in age-damaged proteins as part of an essential protein repair process. Here, we report crystal structures of the repair methyltransferase at resolns. up to 1.2 Å from the hyperthermophilic archaeon Pyrococcus furiosus. Refined structures include binary complexes with the active cofactor AdoMet, its reaction product S-adenosylhomocysteine (AdoHcy), and adenosine. The enzyme places the methyl-donating cofactor in a deep, electrostatically neg. pocket that is shielded from solvent. Across the multiple crystal structures visualized, the presence or absence of the Me group on the cofactor correlates with a significant conformational change in the enzyme in a loop bordering the active site, suggesting a role for motion in catalysis or cofactor exchange. We also report the structure of a ternary complex of the enzyme with adenosine and the methyl-accepting polypeptide substrate VYP(L-isoAsp)HA at 2.1 Å. The substrate binds in a narrow active site cleft with three of its residues in an extended conformation, suggesting that damaged proteins may be locally denatured during the repair process in cells. Manual and computer-based docking studies on different isomers help explain how the enzyme uses steric effects to make the crit. distinction between normal L-aspartyl and age-damaged L-isoaspartyl and D-aspartyl residues. (c) 2001 Academic Press.
  27. 27
    Bensen, D. C., Rodriguez, S., Nix, J., Cunningham, M. L., and Tari, L. W. (2012) Structure of MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) from Vibrio fischeri in complex with substrate UDP-N-acetylglucosamine and the drug fosfomycin. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 68, 382– 385,  DOI: 10.1107/S1744309112006720
    Google Scholar
    27
    Structure of MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) from Vibrio fischeri in complex with substrate UDP-N-acetylglucosamine and the drug fosfomycin
    Bensen, D. C.; Rodriguez, S.; Nix, J.; Cunningham, M. L.; Tari, L. W.
    Acta Crystallographica, Section F: Structural Biology and Crystallization Communications (2012), 68 (4), 382-385CODEN: ACSFCL; ISSN:1744-3091. (International Union of Crystallography)
    The development of new antibiotics is necessitated by the rapid development of resistance to current therapies. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), which catalyzes the first committed step of bacterial peptidoglycan biosynthesis, is a prime candidate for therapeutic intervention. MurA is the target of the antibiotic fosfomycin, a natural product produced by Streptomyces. Despite possessing a high degree of sequence conservation with MurA enzymes from fosfomycin-susceptible organisms, recent microbiol. studies suggest that MurA from Vibrio fischeri (VfiMurA) may confer fosfomycin resistance via a mechanism that is not yet understood. The crystal structure of VfiMurA in a ternary complex with the substrate UDP-N-acetylglucosamine (UNAG) and fosfomycin has been solved to a resoln. of 1.93 Å. Fosfomycin is known to inhibit MurA by covalently binding to a highly conserved cysteine in the active site of the enzyme. A comparison of the title structure with the structure of fosfomycin-susceptible Haemophilus influenzae MurA (PDB entry ) revealed strikingly similar conformations of the mobile substrate-binding loop and clear electron d. for a fosfomycin-cysteine adduct. Based on these results, there are no distinguishing sequence/structural features in VfiMurA that would translate to a diminished sensitivity to fosfomycin. However, VfiMurA is a robust crystallizer and shares high sequence identity with many clin. relevant bacterial pathogens. Thus, it would serve as an ideal system for use in the structure-guided optimization of new antibacterial agents.
  28. 28
    Robinson, N. E. (2002) Protein deamidation. Proc. Natl. Acad. Sci. U. S. A. 99, 5283– 5288,  DOI: 10.1073/pnas.082102799
    Google Scholar
    28
    Protein deamidation
    Robinson, Noah E.
    Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (8), 5283-5288CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    A completely automatic computerized technique for the quant. estn. of the deamidation rates of any protein for which the three-dimensional structure is known has been developed. Calcns. of the specific deamidation rates of 170,014 asparaginyl residues in 13,335 proteins have been carried out. The calcd. values have good quant. reliability when compared with exptl. measurements. These rates demonstrate that deamidation may be a biol. relevant phenomenon in a remarkably large percentage of proteins.
  29. 29
    Dai, S., Lee, D. H., Kenton, N. T., and Zhou, Z. S. (2013) Peptidyl-Asp Metalloendopeptidase. In Handbook of Proteolytic Enzymes 1281– 1285,  DOI: 10.1016/B978-0-12-382219-2.00288-X
    Google Scholar
    There is no corresponding record for this reference.
  30. 30
    Lanzetta, P. A., Alvarez, L. J., Reinach, P. S., and Candia, O. A. (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem. 100, 95– 97,  DOI: 10.1016/0003-2697(79)90115-5
    Google Scholar
    30
    An improved assay for nanomole amounts of inorganic phosphate
    Lanzetta, Peter A.; Alvarez, Lawrence J.; Reinach, Peter S.; Candia, Oscar A.
    Analytical Biochemistry (1979), 100 (1), 95-7CODEN: ANBCA2; ISSN:0003-2697.
    A colorimetric assay for the detn. of nanomol amts. of inorg. phosphate is described. The procedure combines a very high molar absorptivity with color stability and insensitivity to newly released phosphate from labile organophosphates. The quenching procedure of E. E. Baginski et al. (1975) was modified for use with the method of H. H. Hess and J. E. Derr (1975). The color reagent was a mixt. of 0.045% Malachite green-HCl and 4.2% NH4 molybdate in 4N HCl + Sterox. Solns. were read at 660 nm.
  31. 31
    Clarke, J. and Fersht, A. R. (1993) Engineered disulfide bonds as probes of the folding pathway of barnase: increasing the stability of proteins against the rate of denaturation. Biochemistry 32, 4322– 4329,  DOI: 10.1021/bi00067a022
    Google Scholar
    31
    Engineered disulfide bonds as probes of the folding pathway of barnase: Increasing the stability of proteins against the rate of denaturation
    Clarke, Jane; Fersht, Alan R.
    Biochemistry (1993), 32 (16), 4322-9CODEN: BICHAW; ISSN:0006-2960.
    Disulfide bridges have been introduced into barnase to act as probes of folding. One disulfide (between residues 85 and 102) links two loops known to pack together early in the folding pathway. A second disulfide bond (between residues 43 and 80) links two elements of secondary structure known to pack together only after the rate-detg. step of folding. The disulfide-bridged proteins are more stable than wild-type by 4.1 and 1.2 kcal mol-1, resp. The kinetics of unfolding and refolding of the mutant proteins has been measured, and a comparison of the disulfide proteins and their corresponding dithiol forms has been made by use of thermodn. cycles. These data have been used to construct folding profiles of the disulfide proteins. The disulfide bond engineered into the part of the protein that folds early confers stability upon the intermediate and transition states of folding. The protein with a disulfide bond connecting parts of the protein that fold late is not stabilized until the protein reaches its final folded form. Conversely, in the unfolding pathway, the rate of unfolding of this mutant is lowered considerably. This points to a method of decreasing the rate of denaturation of proteins that are used in medical and biotechnol. applications: elements of structure that unfold in or before the rate-detg. step of overall unfolding may be stabilized and so slow down the overall unfolding process. The barnase mutant linked between Cys 43 and Cys 80, for example, unfolds 20 times slower than wild-type and 170 times slower than the reduced protein.
  32. 32
    Fersht, A. (1999) Structure And Mechanism in Protein Science, W. H. Freeman and Company.
    Google Scholar
    There is no corresponding record for this reference.
  33. 33
    Engen, J. R. (2009) Analysis of protein conformation and dynamics by hydrogen/deuterium exchange MS. Anal. Chem. 81, 7870– 7875,  DOI: 10.1021/ac901154s
    Google Scholar
    33
    Analysis of Protein Conformation and Dynamics by Hydrogen/Deuterium Exchange MS
    Engen, John R.
    Analytical Chemistry (Washington, DC, United States) (2009), 81 (19), 7870-7875CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    A review. Understanding as much as possible about proteins in the shortest amt. of time has long been a goal of hydrogen exchange MS [mass spectrometry]. Recent technol. advances in hydrogen exchange MS have led to improvements in the technique, but has this goal yet been achieved.
  34. 34
    Konermann, L., Pan, J., and Liu, Y. H. (2011) Hydrogen exchange mass spectrometry for studying protein structure and dynamics. Chem. Soc. Rev. 40, 1224– 1234,  DOI: 10.1039/C0CS00113A
    Google Scholar
    34
    Hydrogen exchange mass spectrometry for studying protein structure and dynamics
    Konermann, Lars; Pan, Jingxi; Liu, Yu-Hong
    Chemical Society Reviews (2011), 40 (3), 1224-1234CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has become a key technique for monitoring structural and dynamic aspects of proteins in soln. This approach relies on the fact that exposure of a protein to D2O induces rapid amide H D exchange in disordered regions that lack stable hydrogen-bonding. Tightly folded elements are much more protected from HDX, resulting in slow isotope exchange that is mediated by the structural dynamics ("breathing motions") of the protein. MS-based peptide mapping is a well established technique for measuring the mass shifts of individual protein segments. This tutorial review briefly discusses basic fundamentals of HDX/MS, before highlighting a no. of recent developments and applications. Gas phase fragmentation strategies represent a promising alternative to the traditional proteolysis-based approach, but experimentalists have to be aware of scrambling phenomena that can be encountered under certain conditions. Electron-based dissocn. methods provide a soln. to this problem. We also discuss recent advances that facilitate the applicability of HDX/MS to membrane proteins, and to the characterization of short-lived protein folding intermediates. It is hoped that this review will provide a starting point for novices, as well as a useful ref. for practitioners, who require an overview of some recent trends in HDX/MS.
  35. 35
    Martens, C., Shekhar, M., Borysik, A. J., Lau, A. M., Reading, E., Tajkhorshid, E., Booth, P. J., and Politis, A. (2018) Direct protein-lipid interactions shape the conformational landscape of secondary transporters. Nat. Commun. 9, 4151,  DOI: 10.1038/s41467-018-06704-1
    Google Scholar
    35
    Direct protein-lipid interactions shape the conformational landscape of secondary transporters
    Martens Chloe; Borysik Antoni J; Lau Andy M; Reading Eamonn; Booth Paula J; Politis Argyris; Shekhar Mrinal; Tajkhorshid Emad
    Nature communications (2018), 9 (1), 4151 ISSN:.
    Secondary transporters undergo structural rearrangements to catalyze substrate translocation across the cell membrane - yet how such conformational changes happen within a lipid environment remains poorly understood. Here, we combine hydrogen-deuterium exchange mass spectrometry (HDX-MS) with molecular dynamics (MD) simulations to understand how lipids regulate the conformational dynamics of secondary transporters at the molecular level. Using the homologous transporters XylE, LacY and GlpT from Escherichia coli as model systems, we discover that conserved networks of charged residues act as molecular switches that drive the conformational transition between different states. We reveal that these molecular switches are regulated by interactions with surrounding phospholipids and show that phosphatidylethanolamine interferes with the formation of the conserved networks and favors an inward-facing state. Overall, this work provides insights into the importance of lipids in shaping the conformational landscape of an important class of transporters.
  36. 36
    Müller, M. M. (2018) Post-Translational Modifications of Protein Backbones: Unique Functions, Mechanisms, and Challenges. Biochemistry 57, 177– 185,  DOI: 10.1021/acs.biochem.7b00861
    Google Scholar
    36
    Post-Translational Modifications of Protein Backbones: Unique Functions, Mechanisms, and Challenges
    Muller, Manuel M.
    Biochemistry (2018), 57 (2), 177-185CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    A review. Post-translational modifications (PTMs) dramatically enhance the capabilities of proteins. They introduce new functionalities and dynamically control protein activity by modulating intra- and intermol. interactions. Traditionally, PTMs have been considered as reversible attachments to nucleophilic functional groups on amino acid side chains, whereas the polypeptide backbone is often thought to be inert. This paradigm is shifting as chem. and functionally diverse alterations of the protein backbone are discovered. Importantly, backbone PTMs can control protein structure and function just as side chain modifications do, and operate through unique mechanisms to achieve these features. In this perspective, I outline the various types of protein backbone modifications discovered so far and highlight their contributions to biol. as well as the challenges faced in studying these versatile PTMs.
  37. 37
    Mamula, M. J., Gee, R. J., Elliott, J. I., Sette, A., Southwood, S., Jones, P.-J., and Blier, P. R. (1999) Isoaspartyl Post-translational Modification Triggers Autoimmune Responses to Self-proteins. J. Biol. Chem. 274, 22321– 22327,  DOI: 10.1074/jbc.274.32.22321
    Google Scholar
    37
    Isoaspartyl post-translational modification triggers autoimmune responses to self-proteins
    Mamula, Mark J.; Gee, Renelle J.; Elliott, James I.; Sette, Alessandro; Southwood, Scott; Jones, Paul-James; Blier, Peter R.
    Journal of Biological Chemistry (1999), 274 (32), 22321-22327CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The normal functioning immune system is programmed to attack foreign pathogens and other foreign proteins while maintaining tolerance to self-proteins. The mechanisms by which tolerance is broken in the initiation of autoimmunity are not completely understood. In the present study, mice immunized with the murine cytochrome c peptide 90-104 showed no response by the B or T cell compartments. However, immunization with the isoaspartyl form of this peptide, where the linkage of Asp93 to Leu94 occurs through the β-carboxyl group, resulted in strong B and T cell autoimmune responses. Antibodies elicited by immunization with the isoaspartyl form of self-peptide were cross-reactive in binding to both isoforms of cytochrome c peptide and to native cytochrome c self-protein. In a similar manner, immunization of mice with the isoaspartyl form of a peptide autoantigen of human systemic lupus erythematosus (SLE) resulted in strong B and T cell responses while mice maintained tolerance to the normal aspartyl form of self-antigen. Isoaspartyl linkages within proteins are enhanced in aging and stressed cells and arise under physiol. conditions. These post-translationally modified peptides may serve as an early immunol. stimulus in autoimmune disease.
  38. 38
    Manning, M. C., Chou, D. K., Murphy, B. M., Payne, R. W., and Katayama, D. S. (2010) Stability of Protein Pharmaceuticals: An Update. Pharm. Res. 27, 544– 575,  DOI: 10.1007/s11095-009-0045-6
    Google Scholar
    38
    Stability of protein pharmaceuticals: an update
    Manning Mark Cornell; Chou Danny K; Murphy Brian M; Payne Robert W; Katayama Derrick S
    Pharmaceutical research (2010), 27 (4), 544-75 ISSN:.
    In 1989, Manning, Patel, and Borchardt wrote a review of protein stability (Manning et al., Pharm. Res. 6:903-918, 1989), which has been widely referenced ever since. At the time, recombinant protein therapy was still in its infancy. This review summarizes the advances that have been made since then regarding protein stabilization and formulation. In addition to a discussion of the current understanding of chemical and physical instability, sections are included on stabilization in aqueous solution and the dried state, the use of chemical modification and mutagenesis to improve stability, and the interrelationship between chemical and physical instability.
  39. 39
    Yokoyama, H., Mizutani, R., Noguchi, S., and Hayashida, N. (2019) Structural and biochemical basis of the formation of isoaspartate in the complementarity-determining region of antibody 64M-5 Fab. Sci. Rep. 9, 9,  DOI: 10.1038/s41598-019-54918-0
    Google Scholar
    There is no corresponding record for this reference.
  40. 40
    Jefferis, R. (2016) Posttranslational Modifications and the Immunogenicity of Biotherapeutics. J. Immunol. Res. 2016, 5358272,  DOI: 10.1155/2016/5358272
    Google Scholar
    40
    Posttranslational Modifications and the Immunogenicity of Biotherapeutics
    Jefferis Roy
    Journal of immunology research (2016), 2016 (), 5358272 ISSN:.
    Whilst the amino acid sequence of a protein is determined by its gene sequence, the final structure and function are determined by posttranslational modifications (PTMs), including quality control (QC) in the endoplasmic reticulum (ER) and during passage through the Golgi apparatus. These processes are species and cell specific and challenge the biopharmaceutical industry when developing a production platform for the generation of recombinant biologic therapeutics. Proteins and glycoproteins are also subject to chemical modifications (CMs) both in vivo and in vitro. The individual is naturally tolerant to molecular forms of self-molecules but nonself variants can provoke an immune response with the generation of anti-drug antibodies (ADA); aggregated forms can exhibit enhanced immunogenicity and QC procedures are developed to avoid or remove them. Monoclonal antibody therapeutics (mAbs) are a special case because their purpose is to bind the target, with the formation of immune complexes (ICs), a particular form of aggregate. Such ICs may be removed by phagocytic cells that have antigen presenting capacity. These considerations may frustrate the possibility of ameliorating the immunogenicity of mAbs by rigorous exclusion of aggregates from drug product. Alternate strategies for inducing immunosuppression or tolerance are discussed.
  41. 41
    de Brevern, A. G. (2016) Extension of the classical classification of β-turns. Sci. Rep. 6, 33191,  DOI: 10.1038/srep33191
    Google Scholar
    41
    Extension of the classical classification of β-turns
    de Brevern, Alexandre G.
    Scientific Reports (2016), 6 (), 33191CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    The functional properties of a protein primarily depend on its three-dimensional (3D) structure. These properties have classically been assigned, visualized and analyzed on the basis of protein secondary structures. The β-turn is the third most important secondary structure after helixes and β-strands. β-Turns have been classified according to the values of the dihedral angles φ and ψ of the central residue. Conventionally, eight different types of β-turns have been defined, whereas those that cannot be defined are classified as type IV β-turns. This classification remains the most widely used. Nonetheless, the misc. type IV β-turns represent 1/3rd of β-turn residues. An unsupervised specific clustering approach was designed to search for recurrent new turns in the type IV category. The classical rules of β-turn type assignment were central to the approach. The four most frequently occurring clusters defined the new β-turn types. Unexpectedly, these types, designated IV1, IV2, IV3 and IV4, represent half of the type IV β-turns and occur more frequently than many of the previously established types. These types show convincing particularities, in terms of both structures and sequences that allow for the classical β-turn classification to be extended for the first time in 25 years.
  42. 42
    Chatterjee, S., Roy, R. S., and Balaram, P. (2007) Expanding the polypeptide backbone: hydrogen-bonded conformations in hybrid polypeptides containing the higher homologues of alpha-amino acids. J. R. Soc., Interface 4, 587– 606,  DOI: 10.1098/rsif.2006.0203
    Google Scholar
    42
    Expanding the polypeptide backbone: hydrogen-bonded conformations in hybrid polypeptides containing the higher homologues of α-amino acids
    Chatterjee, Sunanda; Roy, Rituparna Sinha; Balaram, P.
    Journal of the Royal Society, Interface (2007), 4 (15), 587-606CODEN: JRSICU; ISSN:1742-5689. (Royal Society)
    A review. Half a century has passed since the hydrogen-bonded secondary structures of polypeptides and proteins were first recognized. An extraordinary wealth of conformational information is now available on peptides and proteins, which are formed of α-amino acid residues. More recently, the discovery of well-folded structures in oligopeptides contg. β-amino acids has focused a great deal of current interest on the conformational properties of peptides constructed from higher homologues (ω) of α-amino acids. This review examines the nature of intramolecularly hydrogen-bonded conformations of hybrid peptides formed by amino acid residues, with a varying no. of backbone atoms. The β-turn, a ubiquitous structural feature formed by two residue (αα) segments in proteins and peptides, is stabilized by a 10-atom (C10) intramol. 4→1 hydrogen bond. Hybrid turns may be classified by comparison with their αα counterparts. The available crystallog. information on hydrogen-bonded hybrid turns is surveyed in this review. Several recent examples demonstrate that individual ω-amino acid residues and hybrid dipeptide segments may be incorporated into the regular structures of α-peptides. Examples of both peptide helixes and hairpins are presented. The present review explores the relationships between folded conformations in hybrid sequences and their counterparts in all α-residue sequences. The use of stereochem. constrained ω-residues promises to expand the range of peptide design strategies to include ω-amino acids. This approach is exemplified by well-folded structures like the C12 (αγ) and C14 (γγ) helixes formed in short peptides contg. multiply substituted γ-residues. The achiral γ-residue gabapentin is a readily accessible building block in the design of peptides contg. γ-amino acids. The construction of globular polypeptide structures using diverse hybrid sequences appears to be a realistic possibility.
  43. 43
    George, K. L. and Horne, W. S. (2018) Foldamer Tertiary Structure through Sequence-Guided Protein Backbone Alteration. Acc. Chem. Res. 51, 1220– 1228,  DOI: 10.1021/acs.accounts.8b00048
    Google Scholar
    43
    Foldamer Tertiary Structure through Sequence-Guided Protein Backbone Alteration
    George, Kelly L.; Horne, W. Seth
    Accounts of Chemical Research (2018), 51 (5), 1220-1228CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review. The prospect of recreating the complex structural hierarchy of protein folding in synthetic oligomers with backbones that are artificial in covalent structure ("foldamers") has long fascinated chemists. Foldamers offer complex functions from biostable scaffolds and have found widespread applications in fields from biomedical to materials science. Most precedent has focused on isolated secondary structures or their assemblies. In considering the goal of complex protein-like tertiary folding patterns, a key barrier became apparent. How does one design a backbone with covalent connectivity and a sequence of side-chain functional groups that will support defined intramol. packing of multiple artificial secondary structures. Two developments were key to overcoming this challenge. First was the recognition of the power of blending α-amino acid residues with monomers differing in backbone connectivity to create "heterogeneous-backbone" foldamers. Second was the finding that replacing some of the natural α-residues in a biol. sequence with artificial-backbone variants can result in a mimic that retains both the fold and function of the native sequence and, in some cases, gains advantageous characteristics. Taken together, these precedents lead to a view of a protein as chem. entity having two orthogonal sequences: a sequence of side-chain functional groups and a sep. sequence of backbone units displaying those functional groups. In this Account, the authors describe the lab's work over the last ∼10 years to leverage the above concept of protein sequence duality in order to develop design principles for constructing heterogeneous-backbone foldamers that adopt complex protein-like tertiary folds. Fundamental to the approach is the utilization of a variety of artificial building blocks (e.g., D-α-residues, Cα-Me-α-residues, N-Me-α-residues, β-residues, γ-residues, δ-residues, polymer segments) in concert, replacing a fraction of α-residues in a given prototype sequence. The authors provide an overview of the state-of-the-art in terms of design principles for choosing substitutions based on consideration of local secondary structure and retention of key side-chain functional groups. The authors survey high-resoln. structures of backbone-modified proteins to illustrate how diverse artificial moieties are accommodated in tertiary fold contexts. The authors detail efforts to elucidate how backbone alteration impacts folding thermodn. and describe how such data informs the development of improved design rules. Collectively, a growing body of results by the lab and others spanning multiple protein systems suggests there is a great deal of plasticity with respect to the backbone chem. structures upon which sequence-encoded tertiary folds can manifest. Moreover, these efforts suggest sequence-guided backbone alteration as a broadly applicable strategy for generating foldamers with complex tertiary folding patterns. The authors conclude by offering some perspective regarding the near future of this field, in terms of unanswered questions, technol. needs, and opportunities for new areas of inquiry.
  44. 44
    Kreitler, D. F., Mortenson, D. E., Forest, K. T., and Gellman, S. H. (2016) Effects of Single alpha-to-beta Residue Replacements on Structure and Stability in a Small Protein: Insights from Quasiracemic Crystallization. J. Am. Chem. Soc. 138, 6498– 6505,  DOI: 10.1021/jacs.6b01454
    Google Scholar
    44
    Effects of Single α-to-β Residue Replacements on Structure and Stability in a Small Protein: Insights from Quasiracemic Crystallization
    Kreitler, Dale F.; Mortenson, David E.; Forest, Katrina T.; Gellman, Samuel H.
    Journal of the American Chemical Society (2016), 138 (20), 6498-6505CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Synthetic peptides that contain backbone modifications but nevertheless adopt folded structures similar to those of natural polypeptides are of fundamental interest and may provide a basis for biomedical applications. Such mols. can, for example, mimic the ability of natural prototypes to bind to specific target macromols. but resist degrdn. by proteases. We have previously shown that oligomers contg. mixts. of α- and β-amino acid residues ("α/β-peptides") can mimic the α-helix secondary structure, and that properly designed α/β-peptides can bind to proteins that evolved to bind to α-helical partners. Here we report fundamental studies that support the long-range goal of extending the α/β approach to tertiary structures. We have evaluated the impact of single α → β modifications on the structure and stability of the small and well-studied villin headpiece subdomain (VHP). The native state of this 35-residue polypeptide contains several α-helical segments packed around a small hydrophobic core. We examd. α → β substitution at four solvent-exposed positions, Asn19, Trp23, Gln26 and Lys30. In each case, both the β3 homolog of the natural α residue and a cyclic β residue were evaluated. All α → β3 substitutions caused significant destabilization of the tertiary structure as measured by variable-temp. CD, although at some of these positions, replacing the β3 residue with a cyclic β residue led to improved stability. Atomic-resoln. structures of four VHP analogs were obtained via quasiracemic crystn. These findings contribute to a fundamental α/β-peptide knowledge-base by confirming that β3-amino acid residues can serve as effective structural mimics of homologous α-amino acid residues within a natural tertiary fold, which should support rational design of functional α/β analogs of natural poly-α-peptides.
  45. 45
    Reinert, Z. E., Lengyel, G. A., and Horne, W. S. (2013) Protein-like tertiary folding behavior from heterogeneous backbones. J. Am. Chem. Soc. 135, 12528– 12531,  DOI: 10.1021/ja405422v
    Google Scholar
    45
    Protein-like Tertiary Folding Behavior from Heterogeneous Backbones
    Reinert, Zachary E.; Lengyel, George A.; Horne, W. Seth
    Journal of the American Chemical Society (2013), 135 (34), 12528-12531CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Because proteins play vital roles in life, much effort has been invested in their mimicry by synthetic agents. One approach is to design unnatural backbone oligomers (foldamers) that fold like natural peptides. Despite success in secondary structure mimicry by such species, protein-like tertiary folds remain elusive. A fundamental challenge underlying this task is the design of a sequence of side chains that will specify a complex tertiary folding pattern on an unnatural backbone. We report here a sequence-based approach to convert a natural protein with a compact tertiary fold to an analog with a backbone composed of ∼20% unnatural building blocks but folding behavior similar to that of the parent protein.
  46. 46
    Horne, W. S., Price, J. L., and Gellman, S. H. (2008) Interplay among side chain sequence, backbone composition, and residue rigidification in polypeptide folding and assembly. Proc. Natl. Acad. Sci. U. S. A. 105, 9151– 9156,  DOI: 10.1073/pnas.0801135105
    Google Scholar
    46
    Interplay among side chain sequence, backbone composition, and residue rigidification in polypeptide folding and assembly
    Horne, W. Seth; Price, Joshua L.; Gellman, Samuel H.
    Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (27), 9151-9156CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The extent to which polypeptide conformation depends on side-chain compn. and sequence has been widely studied, but less is known about the importance of maintaining an α-amino acid backbone. Here, we examine a series of peptides with backbones that feature different repeating patterns of α- and β-amino acid residues but an invariant side-chain sequence. In the pure α-backbone, this sequence corresponds to the previously studied peptide GCN4-pLI, which forms a very stable four-helix bundle quaternary structure. Phys. characterization in soln. and crystallog. structure detn. show that a variety of α/β-peptide backbones can adopt sequence-encoded quaternary structures similar to that of the a prototype. There is a loss in helix bundle stability upon β-residue incorporation; however, stability of the quaternary structure is not a simple function of β-residue content. We find that cyclically constrained β-amino acid residues can stabilize the folds of α/β-peptide GCN4-pLI analogs and restore quaternary structure formation to backbones that are predominantly unfolded in the absence of cyclic residues. Our results show a surprising degree of plasticity in terms of the backbone compns. that can manifest the structural information encoded in a sequence of amino acid side chains. These findings offer a framework for the design of nonnatural oligomers that mimic the structural and functional properties of proteins.
  47. 47
    Lengyel, G. A. and Horne, W. S. (2012) Design strategies for the sequence-based mimicry of side-chain display in protein beta-sheets by alpha/beta-peptides. J. Am. Chem. Soc. 134, 15906– 15913,  DOI: 10.1021/ja306311r
    Google Scholar
    47
    Design Strategies for the Sequence-Based Mimicry of Side-Chain Display in Protein β-Sheets by α/β-Peptides
    Lengyel, George A.; Horne, W. Seth
    Journal of the American Chemical Society (2012), 134 (38), 15906-15913CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The sophistication of folding patterns and functions displayed by unnatural-backbone oligomers has increased tremendously in recent years. Design strategies for the mimicry of tertiary structures seem within reach; however, a general method for the mimicry of sheet segments in the context of a folded protein is an unmet need preventing realization of this goal. Previous work has shown that 1→1 α→β-residue substitutions at cross-strand positions in a hairpin-forming α-peptide sequence can generate an α/β-peptide analog that folds in aq. conditions but with a change in side-chain display relative to the natural sequence; this change would prevent application of single β-residue substitutions in a larger protein. Here, we evaluate four different substitution strategies based on replacement of αα dipeptide segments for the ability to retain both sheet folding encoded by a parent α-peptide sequence as well as nativelike side-chain display in the vicinity of the β-residue insertion point. High-resoln. structure detn. and thermodn. anal. of folding by multidimensional NMR suggest that three of the four designs examd. are applicable to larger proteins.
  48. 48
    Arnold, U., Huck, B. R., Gellman, S. H., and Raines, R. T. (2013) Protein prosthesis: beta-peptides as reverse-turn surrogates. Protein Sci. 22, 274– 279,  DOI: 10.1002/pro.2208
    Google Scholar
    48
    Protein prosthesis: β-peptides as reverse-turn surrogates
    Arnold, Ulrich; Huck, Bayard R.; Gellman, Samuel H.; Raines, Ronald T.
    Protein Science (2013), 22 (3), 274-279CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
    The introduction of non-natural modules could provide unprecedented control over folding/unfolding behavior, conformational stability, and biol. function of proteins. Success requires the interrogation of candidate modules in natural contexts. Here, expressed protein ligation is used to replace a reverse turn in bovine pancreatic RNase (RNase A) with a synthetic β-dipeptide: β2-homoalanine-β3-homoalanine. This segment is known to adopt an unnatural reverse-turn conformation that contains a 10-membered ring hydrogen bond, but one with a donor-acceptor pattern opposite to that in the 10-membered rings of natural reverse turns. The RNase A variant has intact enzymic activity, but unfolds more quickly and has diminished conformational stability relative to native RNase A. These data indicate that hydrogen-bonding pattern merits careful consideration in the selection of beneficial reverse-turn surrogates.
  49. 49
    Arnold, U., Hinderaker, M. P., Nilsson, B. L., Huck, B. R., Gellman, S. H., and Raines, R. T. (2002) Protein prosthesis: a semisynthetic enzyme with a beta-peptide reverse turn. J. Am. Chem. Soc. 124, 8522– 8523,  DOI: 10.1021/ja026114n
    Google Scholar
    49
    Protein prosthesis: A semisynthetic enzyme with a β-peptide reverse turn
    Arnold, Ulrich; Hinderaker, Matthew P.; Nilsson, Bradley L.; Huck, Bayard R.; Gellman, Samuel H.; Raines, Ronald T.
    Journal of the American Chemical Society (2002), 124 (29), 8522-8523CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    β-Amino acids are incorporated into an enzyme by using the method of expressed protein ligation. In the resulting semisynthetic enzyme, an R-nipecotic acid-S-nipecotic acid module replaces Asn113 and Pro114 of RNase A. The semisynthetic enzyme not only retains full catalytic activity but also gains conformational stability. Thus, structural elements can be replaced with foldameric equiv. to endow proteins with more desirable properties.
  50. 50
    Daura, X., Gademann, K., Schäfer, H., Jaun, B., Seebach, D., and van Gunsteren, W. F. (2001) The β-Peptide Hairpin in Solution: Conformational Study of a β-Hexapeptide in Methanol by NMR Spectroscopy and MD Simulation. J. Am. Chem. Soc. 123, 2393– 2404,  DOI: 10.1021/ja003689g
    Google Scholar
    50
    The β-Peptide Hairpin in Solution: Conformational Study of a β-Hexapeptide in Methanol by NMR Spectroscopy and MD Simulation
    Daura, Xavier; Gademann, Karl; Schaefer, Heiko; Jaun, Bernhard; Seebach, Dieter; van Gunsteren, Wilfred F.
    Journal of the American Chemical Society (2001), 123 (10), 2393-2404CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The structural and thermodn. properties of a 6-residue β-peptide I that was designed to form a hairpin conformation have been studied by NMR spectroscopy and mol. dynamics simulation in methanol soln. The predicted hairpin would be characterized by a 10-membered hydrogen-bonded turn involving residues 3 and 4, and two extended antiparallel strands. The interproton distances and backbone torsional dihedral angles derived from the NMR expts. at room temp. are in general terms compatible with the hairpin conformation. Two trajectories of system configurations from 100-ns mol.-dynamics simulations of the peptide in soln. at 298 and 340 K have been analyzed. In both simulations, reversible folding to the hairpin conformation is obsd. Interestingly, there is a significant conformational overlap between the unfolded state of the peptide at each of the temps. As already obsd. in previous studies of peptide folding, the unfolded state is composed of a (relatively) small no. of predominant conformers and in this case lacks any type of secondary-structure element. The trajectories provide an excellent ground for the interpretation of the NMR-derived data in terms of ensemble avs. and distributions as opposed to single-conformation interpretations. From this perspective, a relative population of the hairpin conformation of 20% to 30% would suffice to explain the NMR-derived data. Surprisingly, however, the ensemble of structures from the simulation at 340 K reproduces more accurately the NMR-derived data than the ensemble from the simulation at 298 K, and this point needs further investigation.
  51. 51
    Mayer, C., Muller, M. M., Gellman, S. H., and Hilvert, D. (2014) Building proficient enzymes with foldamer prostheses. Angew. Chem., Int. Ed. 53, 6978– 6981,  DOI: 10.1002/anie.201400945
    Google Scholar
    51
    Building Proficient Enzymes with Foldamer Prostheses
    Mayer, Clemens; Mueller, Manuel M.; Gellman, Samuel H.; Hilvert, Donald
    Angewandte Chemie, International Edition (2014), 53 (27), 6978-6981CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Foldamers are non-natural oligomers that adopt stable conformations reminiscent of those found in proteins. To evaluate the potential of foldameric subunits for catalysis, semisynthetic enzymes contg. foldamer fragments constructed from α- and β-amino acid residues were designed and characterized. Systematic variation of the α→β substitution pattern and types of β-residue afforded highly proficient hybrid catalysts, thus demonstrating the feasibility of expanding the enzyme-engineering toolkit with non-natural backbones.
  52. 52
    Mortenson, D. E., Kreitler, D. F., Thomas, N. C., Guzei, I. A., Gellman, S. H., and Forest, K. T. (2018) Evaluation of beta-Amino Acid Replacements in Protein Loops: Effects on Conformational Stability and Structure. ChemBioChem 19, 604– 612,  DOI: 10.1002/cbic.201700580
    Google Scholar
    52
    Evaluation of β-Amino Acid Replacements in Protein Loops: Effects on Conformational Stability and Structure
    Mortenson, David E.; Kreitler, Dale F.; Thomas, Nicole C.; Guzei, Ilia A.; Gellman, Samuel H.; Forest, Katrina T.
    ChemBioChem (2018), 19 (6), 604-612CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    β-Amino acids have a backbone that is expanded by one carbon atom relative to α-amino acids, and β residues have been investigated as subunits in protein-like mols. that adopt discrete and predictable conformations. Two classes of β residue have been widely explored in the context of generating α-helix-like conformations: β3-amino acids, which are homologous to α-amino acids and bear a side chain on the backbone carbon adjacent to nitrogen, and residues constrained by a five-membered ring, such the one derived from trans-2-aminocyclopentanecarboxylic acid (ACPC). Substitution of α residues with their β3 homologues within an α-helix-forming sequence generally causes a decrease in conformational stability. Use of a ring-constrained β residue, however, can offset the destabilizing effect of α→β substitution. Here we extend the study of α→β substitutions, involving both β3 and ACPC residues, to short loops within a small tertiary motif. We start from previously reported variants of the Pin1 WW domain that contain a two-, three-, or four-residue β-hairpin loop, and we evaluate α→β replacements at each loop position for each variant. By referral to the .vphi.,ψ angles of the native structure, one can choose a stereochem. appropriate ACPC residue. Use of such logically chosen ACPC residues enhances conformational stability in several cases. Crystal structures of three β-contg. Pin1 WW domain variants show that a native-like tertiary structure is maintained in each case.
  53. 53
    Tsien, R. Y. (1998) THE GREEN FLUORESCENT PROTEIN. Annu. Rev. Biochem. 67, 509– 544,  DOI: 10.1146/annurev.biochem.67.1.509
    Google Scholar
    53
    The green fluorescent protein
    Tsien, Roger Y.
    Annual Review of Biochemistry (1998), 67 (), 509-544CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
    A review, with ∼114 refs. In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochem. and cell biol. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resoln. crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiol. indicators, biosensors, and photochem. memories.
  54. 54
    Schwede, T. F., Rétey, J., and Schulz, G. E. (1999) Crystal Structure of Histidine Ammonia-Lyase Revealing a Novel Polypeptide Modification as the Catalytic Electrophile. Biochemistry 38, 5355– 5361,  DOI: 10.1021/bi982929q
    Google Scholar
    54
    Crystal Structure of Histidine Ammonia-Lyase Revealing a Novel Polypeptide Modification as the Catalytic Electrophile
    Schwede, Torsten F.; Retey, Janos; Schulz, Georg E.
    Biochemistry (1999), 38 (17), 5355-5361CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Histidine ammonia-lyase (EC 4.3.1.3) catalyzes the nonoxidative elimination of the α-amino group of histidine and is closely related to the important plant enzyme phenylalanine ammonia-lyase. The crystal structure of histidase from Pseudomonas putida was detd. at 2.1 Å resoln. revealing a homotetramer with D2 symmetry, the mol. center of which is formed by 20 nearly parallel α-helixes. The chain fold, but not the sequence, resembles those of fumarase C and related proteins. The structure shows that the reactive electrophile is a 4-methylidene-imidazole-5-one, which is formed autocatalytically by cyclization and dehydration of residues 142-144 with the sequence Ala-Ser-Gly. With respect to the first dehydration step, this modification resembles the chromophore of the green fluorescent protein. The active center is clearly established by the modification and by mutations. The obsd. geometry allowed us to model the bound substrate at a high confidence level. A reaction mechanism is proposed.
  55. 55
    Kumar, S., Prakash, S., Gupta, K., Dongre, A., Balaram, P., and Balaram, H. (2016) Unexpected functional implication of a stable succinimide in the structural stability of Methanocaldococcus jannaschii glutaminase. Nat. Commun. 7, 12798,  DOI: 10.1038/ncomms12798
    Google Scholar
    55
    Unexpected functional implication of a stable succinimide in the structural stability of Methanocaldococcus jannaschii glutaminase
    Kumar, Sanjeev; Prakash, Sunita; Gupta, Kallol; Dongre, Aparna; Balaram, Padmanabhan; Balaram, Hemalatha
    Nature Communications (2016), 7 (), 12798CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    Protein ageing is often mediated by the formation of succinimide intermediates. These short-lived intermediates derive from asparaginyl deamidation and aspartyl dehydration and are rapidly converted into β-aspartyl or D-aspartyl residues. Here we report the presence of a highly stable succinimide intermediate in the glutaminase subunit of GMP synthetase from the hyperthermophile Methanocaldoccocus jannaschii. By comparing the biophys. properties of the wild-type protein and of several mutants, we show that the presence of succinimide increases the structural stability of the glutaminase subunit. The protein bearing this modification in fact remains folded at 100 °C and in 8 M guanidinium chloride. Mutation of the residue following the reactive asparagine provides insight into the factors that contribute to the hydrolytic stability of the succinimide. Our findings suggest that sequences that stabilize succinimides from hydrolysis may be evolutionarily selected to confer extreme thermal stability.
  56. 56
    Roth, C., Weizenmann, N., Bexten, N., Saenger, W., Zimmermann, W., Maier, T., and Strater, N. (2017) Amylose recognition and ring-size determination of amylomaltase. Sci. Adv. 3, 3,  DOI: 10.1126/sciadv.1601386
    Google Scholar
    There is no corresponding record for this reference.
  57. 57
    Acedo, J. Z., Bothwell, I. R., An, L., Trouth, A., Frazier, C., and van der Donk, W. A. (2019) O-Methyltransferase-Mediated Incorporation of a beta-Amino Acid in Lanthipeptides. J. Am. Chem. Soc. 141, 16790– 16801,  DOI: 10.1021/jacs.9b07396
    Google Scholar
    57
    O-Methyltransferase-Mediated Incorporation of a β-Amino Acid in Lanthipeptides
    Acedo, Jeella Z.; Bothwell, Ian R.; An, Linna; Trouth, Abby; Frazier, Clara; van der Donk, Wilfred A.
    Journal of the American Chemical Society (2019), 141 (42), 16790-16801CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Lanthipeptides represent a large class of cyclic natural products defined by the presence of lanthionine (Lan) and methyllanthionine (MeLan) cross-links. With the advances in DNA sequencing technologies and genome mining tools, new biosynthetic enzymes capable of installing unusual structural features are continuously being discovered. In this study, we investigated an O-methyltransferase that is a member of the most prominent auxiliary enzyme family assocd. with class I lanthipeptide biosynthetic gene clusters. Despite the prevalence of these enzymes, their function has not been established. Herein, we demonstrate that the O-methyltransferase OlvSA encoded in the olv gene cluster from Streptomyces olivaceus NRRL B-3009 catalyzes the rearrangement of a highly conserved aspartate residue to a β-amino acid, isoaspartate, in the lanthipeptide OlvA(BCSA). We elucidated the NMR soln. structure of the GluC-digested peptide, OlvA(BCSA)GluC, which revealed a unique ring topol. comprising four interlocking rings and positions the isoaspartate residue in a solvent exposed loop that is stabilized by a MeLan ring. Gas chromatog.-mass spectrometry anal. further indicated that OlvA(BCSA) contains two DL-MeLan rings and two Lan rings with an unusual LL-stereochem. Lastly, in vitro reconstitution of OlvSA activity showed that it is a leader peptide-independent and S-adenosyl methionine-dependent O-methyltransferase that mediates the conversion of a highly conserved aspartate residue in a cyclic substrate into a succinimide, which is hydrolyzed to generate an Asp or isoAsp contg. peptide. This overall transformation converts an α-amino acid into a β-amino acid in a ribosomally synthesized peptide, via an electrophilic intermediate that may be the intended product.
  58. 58
    Janetzko, J. and Walker, S. (2017) Aspartate Glycosylation Triggers Isomerization to Isoaspartate. J. Am. Chem. Soc. 139, 3332– 3335,  DOI: 10.1021/jacs.6b12866
    Google Scholar
    58
    Aspartate glycosylation triggers isomerization to isoaspartate
    Janetzko, John; Walker, Suzanne
    Journal of the American Chemical Society (2017), 139 (9), 3332-3335CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    O-linked β-N-acetylglucosamine transferase (OGT) is an essential human enzyme that glycosylates numerous nuclear and cytoplasmic proteins on Ser and Thr residues. It also cleaves host cell factor 1 (HCF-1) by a mechanism in which the 1st step involves glycosylation on Glu residues. Replacing Glu with Asp residues in an HCF-1 proteolytic repeat was shown to prevent peptide backbone cleavage, but whether Asp glycosylation occurred was not examd. Here, we report that OGT glycosylates Asp residues much faster than it glycosylates Glu residues in an otherwise identical model peptide substrate; moreover, once formed, the glycosylated Asp residue reacts further to form a succinimide intermediate that hydrolyzes to produce the corresponding isoaspartyl peptide. Asp to isoAsp residue isomerization in proteins occurs in cells, but was previously thought to be exclusively nonenzymic. These findings suggest it may also be enzyme-catalyzed. In addn. to OGT, enzymes that may catalyze Asp to isoAsp isomerization include poly(ADP-ribose) polymerases, enzymes known to ribosylate Asp residues in the process of poly(ADP-ribosyl)ation.
  59. 59
    Deverman, B. E., Cook, B. L., Manson, S. R., Niederhoff, R. A., Langer, E. M., Rosova, I., Kulans, L. A., Fu, X. Y., Weinberg, J. S., Heinecke, J. W., Roth, K. A., and Weintraub, S. J. (2002) Bcl-X-L deamidation is a critical switch in the regulation of the response to DNA damage. Cell 111, 51– 62,  DOI: 10.1016/S0092-8674(02)00972-8
    Google Scholar
    59
    Bcl-xL deamidation is a critical switch in the regulation of the response to DNA damage
    Deverman, Benjamin E.; Cook, Brian L.; Manson, Scott R.; Niederhoff, Robert A.; Langer, Ellen M.; Rosova, Ivana; Kulans, Laura A.; Fu, Xiaoyun; Weinberg, Justin S.; Heinecke, Jay W.; Roth, Kevin A.; Weintraub, Steven J.
    Cell (Cambridge, MA, United States) (2002), 111 (1), 51-62CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    The therapeutic value of DNA-damaging antineoplastic agents is dependent upon their ability to induce tumor cell apoptosis while sparing most normal tissues. Here, we show that a component of the apoptotic response to these agents in several different types of tumor cells is the deamidation of two asparagines in the unstructured loop of Bcl-xL, and we demonstrate that deamidation of these asparagines imports susceptibility to apoptosis by disrupting the ability of Bcl-xL to block the proapoptotic activity of BH3 domain-only proteins. Conversely, Bcl-xL deamidation is actively suppressed in fibroblasts, and suppression of deamidation is an essential component of their resistance to DNA damage-induced apoptosis. Our results suggest that the regulation of Bcl-xL deamidation has a crit. role in the tumor-specific activity of DNA-damaging antineoplastic agents.
  60. 60
    Lee, J. C., Kang, S. U., Jeon, Y., Park, J. W., You, J. S., Ha, S. W., Bae, N., Lubec, G., Kwon, S. H., Lee, J. S., Cho, E. J., and Han, J. W. (2012) Protein L-isoaspartyl methyltransferase regulates p53 activity. Nat. Commun. 3, 3,  DOI: 10.1038/ncomms1933
    Google Scholar
    There is no corresponding record for this reference.
  61. 61
    Curnis, F., Longhi, R., Crippa, L., Cattaneo, A., Dondossola, E., Bachi, A., and Corti, A. (2006) Spontaneous formation of L-isoaspartate and gain of function in fibronectin. J. Biol. Chem. 281, 36466– 36476,  DOI: 10.1074/jbc.M604812200
    Google Scholar
    61
    Spontaneous Formation of L-Isoaspartate and Gain of Function in Fibronectin
    Curnis, Flavio; Longhi, Renato; Crippa, Luca; Cattaneo, Angela; Dondossola, Eleonora; Bachi, Angela; Corti, Angelo
    Journal of Biological Chemistry (2006), 281 (47), 36466-36476CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Isoaspartate formation in extracellular matrix proteins, by aspartate isomerization or asparagine deamidation, is generally viewed as a degrdn. reaction occurring in vivo during tissue aging. For instance, non-enzymic isoaspartate formation at RGD-integrin binding sites causes loss of cell adhesion sites, which in turn can be enzymically "repaired" to RGD by protein-L-isoAsp-O-methyltransferase. We show here that isoaspartate formation is also a mechanism for extracellular matrix activation. In particular, we show that deamidation of Asn263 at the Asn-Gly-Arg (NGR) site in fibronectin N-terminal region generates an αvβ3-integrin binding site contg. the L-isoDGR sequence, which is enzymically "deactivated" to DGR by protein-L-isoAsp-O-methyltransferase. Furthermore, rapid NGR-to-isoDGR sequence transition in fibronectin fragments generates αvβ3 antagonists (named "isonectins") that competitively bind RGD binding sites and inhibit endothelial cell adhesion, proliferation, and tumor growth. Time-dependent generation of isoDGR may represent a sort of mol. clock for activating latent integrin binding sites in proteins.
  62. 62
    Mallagaray, A., Creutznacher, R., Dulfer, J., Mayer, P. H. O., Grimm, L. L., Orduna, J. M., Trabjerg, E., Stehle, T., Rand, K. D., Blaum, B. S., Uetrecht, C., and Peters, T. (2019) A post-translational modification of human Norovirus capsid protein attenuates glycan binding. Nat. Commun. 10, 10,  DOI: 10.1038/s41467-019-09251-5
    Google Scholar
    There is no corresponding record for this reference.
  63. 63
    Lindner, H., Sarg, B., Grunicke, H., and Helliger, W. (1999) Age-dependent deamidation of H1(0) histones in chromatin of mammalian tissues. J. Cancer Res. Clin. Oncol. 125, 182– 186,  DOI: 10.1007/s004320050261
    Google Scholar
    63
    Age-dependent deamidation of H1° histones in chromatin of mammalian tissues
    Lindner, Herbert; Sarg, Bettina; Grunicke, Hans; Helliger, Wilfried
    Journal of Cancer Research and Clinical Oncology (1999), 125 (3/4), 182-186CODEN: JCROD7; ISSN:0171-5216. (Springer-Verlag)
    The compn. of the H1° histone sub-fractions was examd. in rat and mouse tissues. Using reverse-phase HPLC and hydrophilic-interaction liq. chromatog. the authors have found that the relative proportions of all 4 forms of H1° differ from tissue to tissue and from species to species. In principle, the authors obsd. an age-dependent increase in the amt. of both the N-terminally acetylated (H1°a Asn-3 and H1°a Asp-3) and the deamidated forms of H1° (H°a Asp-3 and H1°b Asp-3). Compared with the proportion of N-terminally acetylated H1° forms in liver, kidney, and brain of rats and mice 20 days of age, the authors found an increase in these H1° subfractions of ≤30% in the corresponding organs of 300-day-old animals. The proportion at deamidated H1° forms was 1.6-4-fold higher in the livers and 8-12-fold higher in the brains of 300-day-old mice and rats, resp., than in 20-day-old animals. The tissue-specific nature of the ratio of H1° subfractions suggests that the different forms of histone H1° have specific individual functions. The possible biol. significance of age-related accumulation of N-terminal acetylated and deamidated histone H1° forms is discussed in the light of these results.
  64. 64
    David, C. L., Keener, J., and Aswad, D. W. (1999) Isoaspartate in ribosomal protein S11 of Escherichia coli. J. Bacteriol. 181, 2872– 2877,  DOI: 10.1128/JB.181.9.2872-2877.1999
    Google Scholar
    64
    Isoaspartate in ribosomal protein S11 of Escherichia coli
    David, Cynthia L.; Keener, John; Aswad, Dana W.
    Journal of Bacteriology (1999), 181 (9), 2872-2877CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    Isoaspartyl sites, in which an aspartic acid residue is linked to its C-flanking neighbor via its β-carboxyl side chain, are generally assumed to be an abnormal modification arising as proteins age. The enzyme protein L-isoaspartate methyltransferase (PIMT), present in many bacteria, plants, and animals, catalyzes the conversion of isoaspartate to normal α-linked aspartyl bonds and is thought to serve an important repair function in cells. Having introduced a plasmid into Escherichia coli that allows high-level expression of rat PIMT, we explored the possibility that the rat enzyme reduces isoaspartate levels in E. coli proteins, a result predicted by the repair hypothesis. The present study demonstrates that this is indeed the case; E. coli cells expressing rat PIMT had significantly lower isoaspartate levels than control cells, esp. in stationary phase. Moreover, the distribution of isoaspartate-contg. proteins in E. coli differed dramatically between logarithmic-and stationary-phase cultures. In stationary-phase cells, a no. of proteins in the mol. mass range of 66 to 14 kDa contained isoaspartate, whereas in logarithmic-phase cells, nearly all of the detectable isoaspartate resided in a single 14-kDa protein which we identified as ribosomal protein S11. The near stoichiometric levels of isoaspartate in S11, estd. at 0.5 mol of isoaspartate per mol of S11, suggests that this unusual modification may be important for S11 function.

Cited By

Click to copy section linkSection link copied!
Citation Statements
Explore this article's citation statements on scite.ai
powered by  Scite

This article is cited by 8 publications.

  1. Tianze Zhang, Luis F. Guerra, Yana Berlina, Jon R. Wilson, Beat Fierz, Manuel M. Müller. Semisynthesis of Isomerized Histone H4 Reveals Robustness and Vulnerability of Chromatin toward Molecular Aging. Journal of the American Chemical Society 2025, 147 (6) , 4952-4961. https://doi.org/10.1021/jacs.4c14136
  2. Robert Creutznacher, Eric Schulze-Niemand, Patrick König, Vesna Stanojlovic, Alvaro Mallagaray, Thomas Peters, Matthias Stein, Mario Schubert. Conformational Control of Fast Asparagine Deamidation in a Norovirus Capsid Protein. Biochemistry 2023, 62 (5) , 1032-1043. https://doi.org/10.1021/acs.biochem.2c00656
  3. Qifan Wu, Xiaochen Yang, Ying Wang, Hongqiang Qin, Xun‐Cheng Su, Weimin Xuan. Site‐Selectively Accelerating the Generation of β‐Linked Residue Isoaspartate in Proteins. Angewandte Chemie 2025, https://doi.org/10.1002/ange.202500983
  4. Qifan Wu, Xiaochen Yang, Ying Wang, Hongqiang Qin, Xun‐Cheng Su, Weimin Xuan. Site‐Selectively Accelerating the Generation of β‐Linked Residue Isoaspartate in Proteins. Angewandte Chemie International Edition 2025, https://doi.org/10.1002/anie.202500983
  5. Mark Cornell Manning, Ryan E. Holcomb, Robert W. Payne, Joshua M. Stillahn, Brian D. Connolly, Derrick S. Katayama, Hongcheng Liu, James E. Matsuura, Brian M. Murphy, Charles S. Henry, Daan J. A. Crommelin. Stability of Protein Pharmaceuticals: Recent Advances. Pharmaceutical Research 2024, 41 (7) , 1301-1367. https://doi.org/10.1007/s11095-024-03726-x
  6. Kelly Zhi Qi Zhou, Richard Obexer. Non‐Canonical Amino Acids for Engineering Peptides and Proteins with new Functions. Israel Journal of Chemistry 2024, 296 https://doi.org/10.1002/ijch.202400006
  7. Antoine Danchin. In vivo , in vitro and in silico : an open space for the development of microbe‐based applications of synthetic biology. Microbial Biotechnology 2022, 15 (1) , 42-64. https://doi.org/10.1111/1751-7915.13937
  8. Aparna Vilas Dongre, Sudip Das, Asutosh Bellur, Sanjeev Kumar, Anusha Chandrashekarmath, Tarak Karmakar, Padmanabhan Balaram, Sundaram Balasubramanian, Hemalatha Balaram. Structural basis for the hyperthermostability of an archaeal enzyme induced by succinimide formation. Biophysical Journal 2021, 120 (17) , 3732-3746. https://doi.org/10.1016/j.bpj.2021.07.014
Download PDF
Go to Biochemistry
Get e-Alerts
Get e-Alerts

Biochemistry

Cite this: Biochemistry 2020, 59, 39, 3683–3695
Click to copy citationCitation copied!
https://doi.org/10.1021/acs.biochem.0c00502
Published September 15, 2020

Copyright © 2020 American Chemical Society. This publication is licensed under CC-BY.

Article Views

2982

Altmetric

-

Citations

8
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. Formation of isoaspartate and occurrence in MurA. (a) Mechanism of the spontaneous formation and enzyme-catalyzed repair of isoaspartate (isoAsp). Deamidation of asparagine occurs by nucleophilic attack of the backbone nitrogen atom of the C-flanking amino acid. This process leads to formation of a metastable succinimide intermediate, which hydrolyses to a mixture of Asp and isoAsp linkages. isoAsp can be methylated by PIMT to promote succinimide formation and in turn revert some proportions to Asp (gray arrows). AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine. (b) The essential bacterial enzyme MurA features an isoAsp residue (black) in a hairpin, formed post-translationally from Asn67 (pdb: 1ejc). (3) (c) Sequence alignment of structurally characterized MurA variants. Residues that align with the solvent-facing residues of the β-sheet from the E. cloacae variant are shown in green. The isoAsp-containing hairpin is conserved in Enterobacteriaceae and Vibrionaceae. Alternative hairpin sequences are possible, albeit with a requirement for additional residues. Example β-hairpins with one (Bacillus anthracis, 3sg1, light blue (18)), two (Haemophilus influenzae, 2rl2, salmon (19)), and three (Streptococcus pneumoniae, 5wi5, purple (20)) additional residues were superimposed with the structure from E. cloacae (4e7b, black) and are shown on the right. β-Sheet residues are shown in gray, and side chains are omitted for clarity.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 2

    Figure 2. SUMO-MurAWT contains stoichiometric amounts of isoAsp. (a) Biochemical determination of SUMO-MurAWT isoAsp content via PIMT-dependent methylation. 100 pmol of SUMO-MurAWT or control peptides with or without an isoAsp residue is used as the substrate. AdoHcy is quantified by RP-HPLC upon methylation of SUMO-MurAWT under native conditions using catalytic amounts of PIMT (0.73 μM, red) or after preincubation with 3 M GdmCl and treatment with 14.6 μM PIMT (blue). (b–f) Asn67 is fully deamidated: (b) LC-MS analysis of LysC-digested MurAWT. The peaks at 14.7 (c), 15.0 (d), and 15.4 (e) min correspond to peptide VERXGSVWIDASNVNNFSAPYDLVK (residues 64–88 in MurA), with X being Asn, succinimide, and Asp and isoAsp, respectively. The dashed line in part e represents the scale of the spectra in parts c and d. (f) MSE analysis of the 15.4 min peak from part e. The masses of ions B3 (calcd, 385.22 Da; observed, 385.22 Da) and B4 (calcd, 500.25 Da; observed, 500.25 Da) confirm that X corresponds to an (iso)Asp residue.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 3

    Figure 3. The 3D-structure of MurA facilitates Asn isomerization. (a) Measurement of the isoAsp content of MurAWT* after maturation at 37 °C under native (blue circles) and denaturing (3 M GdmCl, green triangles) conditions. Data points for MurAWT* under native conditions are from two independent measurements. MurA contains two Asn–Gly sequences, expected to isomerize with similar rates under denaturing conditions. The isoAsp content of a peptide corresponding to residues 63–73 (MurA63–73) is shown in pink diamonds. (b) Zoom-in for the initial 24 h period in part a (shaded in gray). (c) Temperature dependence of MurA63–73 deamidation measured by HPLC at 48, 55, 65, and 75 °C. An Arrhenius plot is shown in the inset. (d) Half-lives for isoAsp formation at 37 °C. Error bars represent the 95% CI of the fit from parts a and c.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 4

    Figure 4. Characterization of MurA variants. (a) Monomer fraction (gray, left axis) and yield (red, right axis) for SUMO-MurA variants after production and purification by Ni-NTA and size-exclusion chromatography (error bars: s.d. of four (WT) or three (mutants) biological replicates). (b) isoAsp-quantification for SUMO-MurA variants (error bars: s.d. of three biological replicates). The AdoHcy signals were corrected for the AdoHcy signal observed in a control reaction in the absence of substrate. (c) Relative activity measurement of SUMO-MurA variants (error bars: s.d. of four independent experiments). (d) Saturation kinetics of selected variants (error bars: s.d. of four independent experiments). Activities were recorded with 44 nM MurA variant, 1 mM PEP, and 0.6 mM UDP-GlcNAc unless otherwise stated.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 5

    Figure 5. isoAsp formation improves MurA resilience against stress conditions. (a) Relative recovery of monomeric MurAWT and MurAN67D after incubation at nonoptimal temperatures, pH, and salt concentrations. Standard conditions: 150 mM NaCl, 10 mM Tris, 8 mM sodium phosphate, 2 mM KCl, pH 7.5. High salt: standard conditions with 1.6 M NaCl. Low salt: standard conditions with 40 mM NaCl. Low pH: standard conditions with pH = 6.0–6.2 (error bars: s.d. of three independent experiments). (b) Refolding yield upon partial denaturation with urea. MurAWT and MurAN67D were incubated in 2 M urea for 3 h at room temperature and subsequently dialyzed overnight at 4 °C.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 6

    Figure 6. isoAsp67 impacts the stability of MurA. (a) Chemical denaturation of MurAWT (blue squares) and MurAN67D (green triangles). The fraction unfolded was calculated from the circular dichroism signal at 222 nm. CD signals were recorded in the presence of 0–9 M urea at a protein concentration of 3 μM at 25 °C in a 1 mm cuvette. Unfolding curves are fitted to a two-state model as described in the methods section. (32) Individual data points from three (MurAWT) and four (MurAN67D) experiments are shown after correction for baseline slopes and calculation of the fraction unfolded at each [urea]. (b) Thermal denaturation of MurAWT (blue squares) and MurAN67D (green triangles). A representative plot from two independent measurements is shown. CD signals at 222 nm were recorded at a protein concentration of 1 μM between 4 and 95 °C at 1 °C intervals using a 1 cm cuvette. (c) Slope of the thermal denaturation curve. The first derivative from the curves in part b were calculated after smoothing the denaturation curve with a 2nd degree polynomial and 4 adjacent data points.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 7

    Figure 7. HDX-MS analysis of MurA structure and dynamics. (a) Difference of deuterium uptake between MurAN67D and MurAWT. Woods plots (exposure time sums) from two biological replicates are combined. The dashed line corresponds to a 99% confidence interval. Mutation of isoAsp67 significantly increases the deuterium uptake between residues 41–55 and 55–71. (b) Regions of MurA displaying significantly enhanced deuterium uptake in MurAN67D as compared to MurAWT are colored in red and mapped onto the structure of MurA (pdb: 1ejc). (c–e) Deuterium uptake plots of peptides surrounding residues 67 of MurAWT (blue squares) and MurAN67D (green triangles). The location of the corresponding peptides within the structure of MurA is shown in gold (right panels). Two biological replicates were recorded (solid and dashed lines) with three technical replicates each (error bars: s.d.).

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 8

    Figure 8. isoAsp enables the formation of an unusual β-hairpin. (a) Comparison of the isoAsp-containing β-hairpin of MurA from E. cloacae (4e7b, yellow) with a typical Type II′ turn (1uxa, green). H-bonds are indicated by dashed lines. The backbone atoms of a 12- and 14-membered H-bonded ring are indicated for MurA (NH of isoAsp67 to CO of Ser69) and the Type II′ turn (NH of i to CO of i + 3), respectively. A 10-membered H-bonded ring of the Type II′ turn (CO of i to NH of i + 3) is shown in gray. ChemDraw structures of the turns are shown on the right (MurA, top; Type II′, bottom). (b) The α-carboxylate of isoAsp67 interacts with Lys46 (dashed line, O–N distance = 3.35 Å).

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    This article references 64 other publications.

    1. 1
      Reissner, K. J. and Aswad, D. W. (2003) Deamidation and isoaspartate formation in proteins: unwanted alterations or surreptitious signals?. Cell. Mol. Life Sci. 60, 1281– 1295,  DOI: 10.1007/s00018-003-2287-5
      1
      Deamidation and isoaspartate formation in proteins: Unwanted alterations or surreptitious signals?
      Reissner, K. J.; Aswad, D. W.
      Cellular and Molecular Life Sciences (2003), 60 (7), 1281-1295CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
      A review and discussion. The formation of β-linked Asp-Xaa peptide bonds [isoaspartyl (isoAsp) sites] arise in proteins via succinimide-linked deamidation of Asn or dehydration of Asp residues, reactions which represent a major source of spontaneous protein damage under physiol. conditions. Accumulation of atypical isoAsp sites is minimized in vivo by the activity of protein L-isoaspartyl O-methyltransferase (PIMT), which regenerates a normal peptide bond. The loss of PIMT has harmful consequences, esp. in neurons; thus, formation of isoAsp sites and their subsequent correction by PIMT is widely believed to constitute an important pathway of protein damage and repair. Recent evidence is mounting, however, that deamidation and isoAsp formation may, in some instances, constitute a novel mechanism for intentional modification of protein structure. Here, the authors describe the mechanism of Asx rearrangement, summarize evidence that PIMT serves an important repair function, and then focus on emerging evidence that deamidation and isoAsp formation may sometimes have a useful function.
    2. 2
      Robinson, N. E., and Robinson, A. (2004) Molecular clocks: deamidation of asparaginyl and glutaminyl residues in peptides and proteins, Althouse Press.
      There is no corresponding record for this reference.
    3. 3
      Eschenburg, S. and Schonbrunn, E. (2000) Comparative X-ray analysis of the un-liganded fosfomycin-target MurA. Proteins: Struct., Funct., Genet. 40, 290– 298,  DOI: 10.1002/(SICI)1097-0134(20000801)40:2<290::AID-PROT90>3.0.CO;2-0
      3
      Comparative X-ray analysis of the un-liganded fosfomycin-target MurA
      Eschenburg, Susanne; Schonbrunn, Ernst
      Proteins: Structure, Function, and Genetics (2000), 40 (2), 290-298CODEN: PSFGEY; ISSN:0887-3585. (Wiley-Liss, Inc.)
      MurA, an essential enzyme for the synthesis of the bacterial cell wall, follows an induced-fit mechanism. Upon substrate binding, the active site forms in the interdomain cleft, involving movements of the two domains of the protein and a reorientation of the loop Pro112-Pro121. We compare two structures of un-liganded MurA from Enterobacter cloacae: a new orthorhombic form, solved to 1.80 Å resoln., and a monoclinic form, redetd. to 1.55 Å resoln. In the monoclinic form, the loop Pro112-Pro121 stretches into solvent, while in the new form it adopts a wound conformation, thereby reducing solvent accessibility of the crit. residue Cys115. In the interdomain cleft a network of 27 common water mols. has been identified, which partially shields neg. charges in the cleft and stabilizes the orientation of catalytically crucial residues. This could support substrate binding and ease domain movements. Near the hinge region an isoaspartyl residue has been recognized, which is the product of post-translational modification of the genetically encoded Asn67-Gly68. The homogeneous population with L-isoaspartate in both structures suggests that the modification in Enterobacter cloacae MurA is not a mere aging defect but rather the result of a specific in vivo process.
    4. 4
      Robinson, N. E. and Robinson, A. B. (2001) Molecular clocks. Proc. Natl. Acad. Sci. U. S. A. 98, 944– 949,  DOI: 10.1073/pnas.98.3.944
      4
      Molecular clocks
      Robinson, Noah E.; Robinson, Arthur B.
      Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (3), 944-949CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      A convenient and precise mass spectrometric method for measurement of the deamidation rates of glutaminyl and asparaginyl residues in peptides and proteins has been developed; the rates of deamidation of 306 asparaginyl sequences in model peptides at pH 7.4, 37.0°, 0.15 M Tris·HCl buffer have been detd.; a library of 913 amide-contg. peptides for use by other investigators in similar studies has been established; and, by means of simultaneous deamidation rate measurements of rabbit muscle aldolase and appropriate model peptides in the same solns., the use of this method for quant. measurement of the relative effects of primary, secondary, tertiary, and quaternary protein structure on deamidation rates has been demonstrated. The measured rates are discussed with respect to the hypothesis that glutaminyl and asparaginyl residues serve, through deamidation, as mol. timers of biol. events.
    5. 5
      Xie, M. and Schowen, R. L. (1999) Secondary Structure and Protein Deamidation. J. Pharm. Sci. 88, 8– 13,  DOI: 10.1021/js9802493
      5
      Secondary Structure and Protein Deamidation
      Xie, Minli; Schowen, Richard L.
      Journal of Pharmaceutical Sciences (1999), 88 (1), 8-13CODEN: JPMSAE; ISSN:0022-3549. (American Chemical Society)
      The deamidation reactions of asparagine residues in α-helical and β-turn secondary structural environments of peptides and proteins are reviewed with 31 refs. Both kinds of secondary structure tend to stabilize asparagine residues against deamidation, although the effects are not large. The effect of β-sheet structures on asparagine stability is unclear, although simple considerations suggest a stabilization in this environment also.
    6. 6
      Shimizu, T., Matsuoka, Y., and Shirasawa, T. (2005) Biological Significance of Isoaspartate and Its Repair System. Biol. Pharm. Bull. 28, 1590– 1596,  DOI: 10.1248/bpb.28.1590
      6
      Biological significance of isoaspartate and its repair system
      Shimizu, Takahiko; Matsuoka, Yasuji; Shirasawa, Takuji
      Biological & Pharmaceutical Bulletin (2005), 28 (9), 1590-1596CODEN: BPBLEO; ISSN:0918-6158. (Pharmaceutical Society of Japan)
      A review. The isomerization of L-aspartate and the deamidation of L-asparagine in proteins or peptides dominantly give rise to L-isoaspartate by a nonenzymic reaction via succinimide as a intermediate under physiol. conditions. Isoaspartates have been identified in a variety of cellular proteins in vivo as well as pathol. deposited proteins in neurodegenerative brain tissue. Here, the authors describe the enhanced formation of isoaspartate in amyloid-β (Aβ) peptides in Alzheimer's disease (AD). Specific antibodies recognizing isoaspartate in Aβ have revealed that isomerized Aβ peptides are deposited in senile plaques as well as amyloid-bearing vessels. Moreover, it has been obsd. that Aβ peptides, isomerized at positions 7 or 23, are differentially deposited in senile plaques and vascular amyloids in AD brains. In vitro expts. have shown that the modification at position 23 greatly enhances the aggregation of Aβ. Furthermore, systematic proline substitution analyses has revealed that the β-turn structure at positions 22 and 23 of Aβ42 plays a crucial role in the aggregation and neurotoxicity of Aβ peptides. It is suggested that spontaneous isomerization at position 23 induces the conformational change to form a β-turn at position 23, which plays a pathogenic role in the deposition of Aβ peptides in sporadic AD. Protein L-isoaspartyl methyltransferase (PIMT) is a putative protein repair enzyme, which converts L-isoaspartyl residues in damaged proteins to normal L-aspartyl residues. PIMT-deficient mice have shown neurodegenerative changes concomitant with the accumulation of L-isoaspartate in the brain. The authors discuss the pathol. implications of the formation of isoaspartate in damaged proteins during neurodegeneration in model mice and AD.
    7. 7
      Fukuda, H., Shimizu, T., Nakajima, M., Mori, H., and Shirasawa, T. (1999) Synthesis, aggregation, and neurotoxicity of the alzheimer’s Aß1–42 amyloid peptide and its isoaspartyl isomers. Bioorg. Med. Chem. Lett. 9, 953– 956,  DOI: 10.1016/S0960-894X(99)00121-3
      7
      Synthesis, aggregation, and neurotoxicity of the Alzheimer's Aβ1-42 amyloid peptide and its isoaspartyl isomers
      Fukuda, Hiroyuki; Shimizu, Takahiko; Nakajima, Mitsunari; Mori, Hiroshi; Shirasawa, Takuji
      Bioorganic & Medicinal Chemistry Letters (1999), 9 (7), 953-956CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)
      Amyloid Aβ1-42 peptide (Aβ1-42) and its isomers with an isoaspartyl residue at position 7 or 23 [Aβ1-42(isoAsp7) and Aβ1-42(isoAsp23)] were synthesized in high purity by the Fmoc-solid phase technique, followed by HPLC on a silica-based reversed-phase column under the basic conditions. Aβ1-42(isoAsp23) aggregated more strongly than native Aβ1-42 and showed significant neurotoxicity, while the aggregation ability and neurotoxicity of Aβ1-42(isoAsp7) was weak. This suggests that the isomerization of the aspartyl residues plays an important role in fibril formation in Alzheimer's disease.
    8. 8
      Shimizu, T., Watanabe, A., Ogawara, M., Mori, H., and Shirasawa, T. (2000) Isoaspartate formation and neurodegeneration in Alzheimer’s disease. Arch. Biochem. Biophys. 381, 225– 234,  DOI: 10.1006/abbi.2000.1955
      8
      Isoaspartate formation and neurodegeneration in Alzheimer's disease
      Shimizu, Takahiko; Watanabe, Atsushi; Ogawara, Midori; Mori, Hiroshi; Shirasawa, Takuji
      Archives of Biochemistry and Biophysics (2000), 381 (2), 225-234CODEN: ABBIA4; ISSN:0003-9861. (Academic Press)
      A review with 99 refs. We reviewed here that protein isomerization is enhanced in amyloid-β peptides (Aβ) and paired helical filaments (PHFs) purified from Alzheimer's disease (AD) brains. Biochem. analyses revealed that Aβ purified from senile plaques and vascular amyloid are isomerized at Asp-1 and Asp-7. A specific antibody recognizing isoAsp-23 of Aβ further suggested the isomerization of Aβ at Asp-23 in vascular amyloid as well as in the core of senile plaques. Biochem. analyses of purified PHFs also revealed that heterogeneous mol. wt. tau contains L-isoaspartate at Asp-193, Asn-381, and Asp-387, indicating a modification, other than phosphorylation, that differentiates between normal tau and PHF tau. Since protein isomerization as L-isoaspartate causes structural changes and functional inactivation, or enhances the aggregation process, this modification is proposed as one of the progression factors in AD. Protein L-isoaspartyl methyltransferase (PIMT) is suggested to play a role in the repair of isomerized proteins contg. L-isoaspartate. We show here that PIMT is upregulated in neurodegenerative neurons and colocalizes in neurofibrillary tangles (NFTs) in AD. Taken together with the enhanced protein isomerization in AD brains, it is implicated that the upregulated PIMT may assoc. with increased protein isomerization in AD. We also reviewed studies on PIMT-deficient mice that confirmed that PIMT plays a physiol. role in the repair of isomerized proteins contg. L-isoaspartate. The knockout study also suggested that the brain of PIMT-deficient mice manifested neurodegenerative changes concomitant with accumulation of L-isoaspartate. We discuss the pathol. implications of protein isomerization in the neurodegeneration found in model mice and AD. (c) 2000 Academic Press.
    9. 9
      Warmack, R. A., Boyer, D. R., Zee, C.-T., Richards, L. S., Sawaya, M. R., Cascio, D., Gonen, T., Eisenberg, D. S., and Clarke, S. G. (2019) Structure of amyloid-β (20–34) with Alzheimer’s-associated isomerization at Asp23 reveals a distinct protofilament interface. Nat. Commun. 10, 3357,  DOI: 10.1038/s41467-019-11183-z
      9
      Structure of amyloid-β (20-34) with Alzheimer's-associated isomerization at Asp23 reveals a distinct protofilament interface
      Warmack Rebeccah A; Boyer David R; Zee Chih-Te; Richards Logan S; Sawaya Michael R; Cascio Duilio; Eisenberg David S; Clarke Steven G; Sawaya Michael R; Cascio Duilio; Gonen Tamir; Eisenberg David S; Clarke Steven G; Sawaya Michael R; Cascio Duilio; Eisenberg David S; Gonen Tamir; Eisenberg David S; Gonen Tamir; Eisenberg David S; Gonen Tamir
      Nature communications (2019), 10 (1), 3357 ISSN:.
      Amyloid-β (Aβ) harbors numerous posttranslational modifications (PTMs) that may affect Alzheimer's disease (AD) pathogenesis. Here we present the 1.1 ÅA resolution MicroED structure of an Aβ 20-34 fibril with and without the disease-associated PTM, L-isoaspartate, at position 23 (L-isoAsp23). Both wild-type and L-isoAsp23 protofilaments adopt β-helix-like folds with tightly packed cores, resembling the cores of full-length fibrillar Aβ structures, and both self-associate through two distinct interfaces. One of these is a unique Aβ interface strengthened by the isoaspartyl modification. Powder diffraction patterns suggest a similar structure may be adopted by protofilaments of an analogous segment containing the heritable Iowa mutation, Asp23Asn. Consistent with its early onset phenotype in patients, Asp23Asn accelerates aggregation of Aβ 20-34, as does the L-isoAsp23 modification. These structures suggest that the enhanced amyloidogenicity of the modified Aβ segments may also reduce the concentration required to achieve nucleation and therefore help spur the pathogenesis of AD.
    10. 10
      Lampi, K. J., Wilmarth, P. A., Murray, M. R., and David, L. L. (2014) Lens β-crystallins: the role of deamidation and related modifications in aging and cataract. Prog. Biophys. Mol. Biol. 115, 21– 31,  DOI: 10.1016/j.pbiomolbio.2014.02.004
      10
      Lens β-crystallins: The role of deamidation and related modifications in aging and cataract
      Lampi, Kirsten J.; Wilmarth, Phillip A.; Murray, Matthew R.; David, Larry L.
      Progress in Biophysics & Molecular Biology (2014), 115 (1), 21-31CODEN: PBIMAC; ISSN:0079-6107. (Elsevier Ltd.)
      A review. Crystallins are the major proteins in the lens of the eye and function to maintain transparency of the lens. Of the human crystallins, α, β, and γ, the β-crystallins remain the most elusive in their structural significance due to their greater no. of subunits and possible oligomer formations. The β-crystallins are also heavily modified during aging. This review focuses on the functional significance of deamidation and the related modifications of racemization and isomerization, the major modifications in β-crystallins of the aged human lens. Elucidating the role of these modifications in cataract formation has been slow, because they are anal. among the most difficult post-translational modifications to study. Recent results suggest that many amides deamidate to similar extent in normal aged and cataractous lenses, while others may undergo greater deamidation in cataract. Mimicking deamidation at crit. structural regions induces structural changes that disrupt the stability of the β-crystallins and lead to their aggregation in vitro. Deamidations at the surface disrupt interactions with other crystallins. Addnl., the α-crystallin chaperone is unable to completely prevent deamidated β-crystallins from insolubilization. Therefore, deamidation of β-crystallins may enhance their pptn. and light scattering in vivo contributing to cataract formation.Future expts. are needed to quantify differences in deamidation rates at all Asn and Gln residues within crystallins from aged and cataractous lenses, as well as racemization and isomerization which potentially perturb protein structure greater than deamidation alone. Quant. data is greatly needed to investigate the importance of these major age-related modifications in cataract formation.
    11. 11
      McFadden, P. N. and Clarke, S. (1987) Conversion of isoaspartyl peptides to normal peptides: implications for the cellular repair of damaged proteins. Proc. Natl. Acad. Sci. U. S. A. 84, 2595– 2599,  DOI: 10.1073/pnas.84.9.2595
      11
      Conversion of isoaspartyl peptides to normal peptides: implications for the cellular repair of damaged proteins
      McFadden, Philip N.; Clarke, Steven
      Proceedings of the National Academy of Sciences of the United States of America (1987), 84 (9), 2595-9CODEN: PNASA6; ISSN:0027-8424.
      The hypothesis that cellular protein carboxymethylation reactions recognize altered aspartyl residues as part of a protein repair pathway has been tested in an in vitro system using tetragastrin (Trp-Met-Asp-Phe-NH2) as a model sequence. The L-isoaspartyl form of tetragastrin, where the phenylalanine residue is linked to the side-chain CO2H group of the aspartate residue ([iso-Asp3]tetragastrin), is a substrate for the erythrocyte protein carboxyl methyltransferases, whereas the normal form is not. The enzymically produced α-Me ester of [iso-Asp3]tetragastrin, [iso-Asp(OMe)3]tetragastrin, is unstable at pH 7.4 and 37° and spontaneously demethylates with a half-time of 41 min to an intermediate L-succinimide form ([Asu3]tetragastrin) that, in turn, spontaneously hydrolyzes with a half-time of 116 min to give a mixt. of normal tetragastrin (20%) and [iso-Asp3]tetragastrin (80%). This sequence of enzymic and nonenzymic reactions can be coupled in a single reaction mixt.; the [iso-Asp3]tetragastrin that is produced upon succinimide hydrolysis can reenter the reaction sequence by enzymic methylation, and the net result of the process is the conversion of the isomerized peptide to the normal peptide. The efficiency of this repair reaction is limited by a side reaction of racemization at the α-C of the succinimide (half-time = 580 min). In a 24-h time period, normal L-aspartyl-contg. tetragastrin is obtained in ∼50% yield from the coupled reaction mixt.; other products include [D-iso-Asp3]tetragastrin and [D-Asp3]tetragastrin. The versatile chem. of succinimide peptides suggests that methylated L-isoaspartyl sites (and possibly methylated D-aspartyl sites) in cellular polypeptides can eventually yield repaired normal L-aspartyl sites through succinimide intermediates.
    12. 12
      Johnson, B. A., Murray, E. D., Clarke, S., Glass, D. B., and Aswad, D. W. (1987) Protein carboxyl methyltransferase facilitates conversion of atypical L-isoaspartyl peptides to normal L-aspartyl peptides. J. Biol. Chem. 262, 5622– 5629
      12
      Protein carboxyl methyltransferase facilitates conversion of atypical L-isoaspartyl peptides to normal L-aspartyl peptides
      Johnson, Brett A.; Murray, E. David, Jr.; Clarke, Steven; Glass, David B.; Aswad, Dana W.
      Journal of Biological Chemistry (1987), 262 (12), 5622-9CODEN: JBCHA3; ISSN:0021-9258.
      Prolonged incubation of L-isoaspartate (L-iso-Asp)-contg. forms of lactate dehydrogenase (231-242), sperm activating peptide, and ACTH (22-27) at 37°, pH 7.4, with S-adenosyl-L-methionine and protein carboxyl methyltransferase (I) from bovine brain leads to extensive conversion of the atypical isopeptide bond to a normal peptide bond. For the lactate dehydrogenase-related peptide, conversion was 80% complete after 24 h. For the other 2 peptides, conversion reached a level of ∼65% after 48 h. The mechanism of conversion involved (1) rapid enzymic methylation of the α-CO2H of the L-iso-Asp residue; (2) nonenzymic demethylation resulting in formation of an L-aspartyl cyclic imide; and (3) a slow, nonenzymic hydrolysis of the cyclic imide to form a mixt. of 15-25% normal L-Asp (aspartate) peptide and 75-85% L-iso-Asp peptide. The regenerated L-iso-Asp peptide was remethylated and the cycle was repeated. The extent of conversion was limited by a competing side reaction wherein the L-imide slowly racemizes, leading to the formation of mainly D-iso-Asp peptide, which was not a substrate for I. The ability of I to initiate conversion of L-iso-Asp residues to normal L-Asp suggests a possible role for this enzyme in facilitating the repair or degrdn. of deamidated proteins in vivo.
    13. 13
      Lowenson, J. D., Kim, E., Young, S. G., and Clarke, S. (2001) Limited Accumulation of Damaged Proteins in L-Isoaspartyl (D-Aspartyl) O-Methyltransferase-deficient Mice. J. Biol. Chem. 276, 20695– 20702,  DOI: 10.1074/jbc.M100987200
      13
      Limited accumulation of damaged proteins in L-isoaspartyl (D-aspartyl) O-methyltransferase-deficient mice
      Lowenson, Jonathan D.; Kim, Edward; Young, Stephen G.; Clarke, Steven
      Journal of Biological Chemistry (2001), 276 (23), 20695-20702CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      L-Isoaspartyl (D-aspartyl) O-methyltransferase (PCMT1) can initiate the conversion of damaged aspartyl and asparaginyl residues to normal L-aspartyl residues. Mice lacking this enzyme (Pcmt1 -/- mice) have elevated levels of damaged residues and die at a mean age of 42 days from massive tonic-clonic seizures. To extend the lives of the knockout mice so that the long term effects of damaged residue accumulation could be investigated, we produced transgenic mice with a mouse Pcmt1 cDNA under the control of a neuron-specific promoter. Pcmt1 transgenic mice that were homozygous for the endogenous Pcmt1 knockout mutation ("transgenic Pcmt1 -/- mice") had brain PCMT1 activity levels that were 6.5-13% those of wild-type mice but had little or no activity in other tissues. The transgenic Pcmt1 -/- mice lived, on av., 5-fold longer than nontransgenic Pcmt1 -/- mice and accumulated only half as many damaged aspartyl residues in their brain proteins. The concn. of damaged residues in heart, testis, and brain proteins in transgenic Pcmt1 -/- mice initially increased with age but unexpectedly reached a plateau by 100 days of age. Urine from Pcmt1 -/- mice contained increased amts. of peptides with damaged aspartyl residues, apparently enough to account for proteins that were not repaired intracellularly. In the absence of PCMT1, proteolysis may limit the intracellular accumulation of damaged proteins but less efficiently than in wild-type mice having PCMT1-mediated repair.
    14. 14
      Yamamoto, A., Takagi, H., Kitamura, D., Tatsuoka, H., Nakano, H., Kawano, H., Kuroyanagi, H., Yahagi, Y.-I., Kobayashi, S.-I., Koizumi, K.-I., Sakai, T., Saito, K.-I., Chiba, T., Kawamura, K., Suzuki, K., Watanabe, T., Mori, H., and Shirasawa, T. (1998) Deficiency in Protein Isoaspartyl Methyltransferase Results in a Fatal Progressive Epilepsy. J. Neurosci. 18, 2063– 2074,  DOI: 10.1523/JNEUROSCI.18-06-02063.1998
      14
      Deficiency in protein L-isoaspartyl methyltransferase results in a fatal progressive epilepsy
      Yamamoto, Akihiro; Takagi, Hideyuki; Kitamura, Daisuke; Tatsuoka, Hozumi; Nakano, Hirotake; Kawano, Hitoshi; Kuroyanagi, Hidehito; Yahagi, Yu-Ichi; Kobayashi, Shin-Ichiro; Koizumi, Ken-Ichi; Sakai, Tsuyoshi; Saito, Ken-Ichi; Chiba, Tanemichi; Kawamura, Koki; Suzuki, Katsushi; Watanabe, Takeshi; Mori, Hiroshi; Shirasawa, Takuji
      Journal of Neuroscience (1998), 18 (6), 2063-2074CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
      Protein L-isoaspartyl methyltransferase (PIMT) is suggested to play a role in the repair of aged protein spontaneously incorporated with isoaspartyl residues. The authors generated PIMT-deficient mice by targeted disruption of the PIMT gene to elucidate the biol. role of the gene in vivo. PIMT-deficient mice died from progressive epileptic seizures with grand mal and myoclonus between 4 and 12 wk of age. An anticonvulsive drug, dipropylacetic acid (DPA), improved their survival but failed to cure the fatal outcome. L-Isoaspartate, the putative substrate for PIMT, was increased ninefold in the brains of PIMT-deficient mice. The brains of PIMT-deficient mice started to enlarge after 4 wk of age when the apical dendrites of pyramidal neurons in cerebral cortices showed aberrant arborizations with disorganized microtubules. The authors conclude that methylation of modified proteins with isoaspartyl residues is essential for the maintenance of a mature CNS and that a deficiency in PIMT results in fatal progressive epilepsy in mice.
    15. 15
      Heijenoort, J. V. (2001) Recent advances in the formation of the bacterial peptidoglycan monomer unit. Nat. Prod. Rep. 18, 503– 519,  DOI: 10.1039/a804532a
      There is no corresponding record for this reference.
    16. 16
      Seebach, D., Beck, A. K., and Bierbaum, D. J. (2004) The World of β- and γ-Peptides Comprised of Homologated Proteinogenic Amino Acids and Other Components. Chem. Biodiversity 1, 1111– 1239,  DOI: 10.1002/cbdv.200490087
      16
      The world of β- and γ-peptides comprised of homologated proteinogenic amino acids and other components
      Seebach, Dieter; Beck, Albert K.; Bierbaum, Daniel J.
      Chemistry & Biodiversity (2004), 1 (8), 1111-1239CODEN: CBHIAM; ISSN:1612-1872. (Verlag Helvetica Chimica Acta AG)
      A review. The origins of our nearly ten-year research program of chem. and biol. investigations into peptides based on homologated proteinogenic amino acids are described. The road from the biopolymer poly[ethyl (R)-3-hydroxybutanoate] to the β-peptides was primarily a step from org. synthesis methodol. (the prepn. of enantiomerically pure compds. (EPCs)) to supramol. chem. (higher-order structures maintained through non-covalent interactions). The performing of biochem. and biol. tests on the β-and γ-peptides, which differ from natural peptides/proteins by a single or two addnl. CH2 groups per amino acid, then led into bioorg. chem. and medicinal chem. The individual chapters of this review article begin with descriptions of work on β-amino acids, β-peptides, and polymers (Nylon-3) that dates back to the 1960s, even to the times of Emil Fischer, but did not yield insights into structures or biol. properties. The numerous, often highly physiol. active, or even toxic, natural products contg. β- and γ-amino acid moieties are then presented. Chapters on the prepn. of homologated amino acids with proteinogenic side chains, their coupling to provide the corresponding peptides, both in soln. (including thioligation) and on the solid phase, their isolation by preparative HPLC, and their characterization by mass spectrometry (HR-MS and MS sequencing) follow. After that, their structures, predominantly detd. by NMR spectroscopy in methanolic soln., are described: helixes, pleated sheets, and turns, together with stack-, crankshaft-, paddlewheel-, and staircase-like patterns. The presence of the addnl. C-C bonds in the backbones of the new peptides did not give rise to a chaotic increase in their secondary structures as many protein specialists might have expected: while there are indeed more structure types than are obsd. in the α-peptide realm - three different helixes (10/12-, 12-, and 14-helix) if we include oligomers of trans-2-aminocyclopentanecarboxylic acid, for example - the structures are already observable with chains made up of only four components, and, having now undergone a learning process, we are able to construct them by design. The structures of the shorter β-peptides can also be reliably detd. by mol.-dynamics calcns. (in soln.; GROMOS program package). Unlike in the case of the natural helixes, these compds.' folding into secondary structures is not cooperative. In β- and γ-peptides, it is possible to introduce heteroatom substituents (such as halogen or OH) onto the backbones or to incorporate heteroatoms (NH, O) directly into the chain, and, thanks to this, it has been possible to study effects unobservable in the world of the α-peptides. Tests with proteolytic enzymes of all types (from mammals, microorganisms, yeasts) and in vivo examn. (mice, rats, insects, plants) showed β- and γ-peptides to be completely stable towards proteolysis and, as demonstrated for two β-peptides, extraordinarily stable towards metab., even when bearing functionalized side chains (such as those of Thr, Tyr, Trp, Lys, or Arg). The β-peptides so far examd. also normally display no or only very weak cytotoxic, antiproliferative, antimicrobial, hemolytic, immunogenic, or inflammatory properties either in cell cultures or in vivo. Even biol. degrdn. by microbial colonies of the types found in sewage-treatment plants or in soil is very slow. Besides these less sharply definable interactions, it has also been possible to construct β- and γ-peptide agonists of naturally occurring peptide hormones, MHC-binding β-peptides, or amphipathic β-peptide inhibitors of membrane-bound proteins in a controlled fashion. Examples include somatostatin mimics and the suppression of cholesterol transport through the intestinal brush-border membrane (by the SR-BI-protein). The results so far obtained from investigations into peptides made up of homologues of the proteinogenic amino acids also represent a contribution to deepening of our knowledge of the natural peptides/proteins, while potential for biomedicinal application of this new class of substances has also been suggested.
    17. 17
      Gellman, S. H. (1998) Foldamers: A manifesto. Acc. Chem. Res. 31, 173– 180,  DOI: 10.1021/ar960298r
      17
      Foldamers: A Manifesto
      Gellman, Samuel H.
      Accounts of Chemical Research (1998), 31 (4), 173-180CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review with 79 refs. on the indentification of new polymeric systems, termed foldamers, with strong tendencies to adopt specific compact conformations, including the development of predictively useful understanding of the relationship between the repetitive features of the monomer structure and conformational properties at the polymer level. Topics include background, case studies of β-peptides, DNA and RNA analogs, and other foldamers.
    18. 18
      Minasov, G., Halavaty, A., Filippova, E. V., Shuvalova, L., Dubrovska, I., Winsor, J., Papazisi, L., and Anderson, W. F. (2011) 2.6 Angstrom Crystal Structure of UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1 (MurA1) from Bacillus anthracis. Worldwide Protein Data Bank 3SG1,  DOI: 10.2210/pdb3SG1/pdb
      There is no corresponding record for this reference.
    19. 19
      Yoon, H. J., Lee, S. J., Mikami, B., Park, H. J., Yoo, J., and Suh, S. W. (2008) Crystal structure of UDP-N-acetylglucosamine enolpyruvyl transferase from Haemophilus influenzae in complex with UDP-N-acetylglucosamine and fosfomycin. Proteins: Struct., Funct., Genet. 71, 1032– 1037,  DOI: 10.1002/prot.21959
      19
      Crystal structure of UDP-N-acetylglucosamine enolpyruvyl transferase from Haemophilus influenzae in complex with UDP-N-acetylglucosamine and fosfomycin
      Yoon, Hye-Jin; Lee, Sang Jae; Mikami, Bunzo; Park, Hyun-Ju; Yoo, Jakyung; Suh, Se Won
      Proteins: Structure, Function, and Bioinformatics (2008), 71 (2), 1032-1037CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
      Peptidoglycan (also known as murein) serves a structural role in the bacterial cell wall. The bacterial enzyme UDP-N-acetylglucosamine enolpyruyl transferase (MurA; also called MurZ; E.C. 2.5.1.7) catalyzes the first committed step of peptidoglycan biosynthesis, that is transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine to form enolpyruyl-UDP-N-acetylglucosamine. We have initiated structure detn. of the 424-residue MurA from H. influenzae. Here we report two crystal structures of H. influenzae MurA: a binary complex with the substrate UDP-N-acetylglucosamine at 2.2 Å resoln. and a ternary complex with UDP-N-acetylglucosamine and fosfomycin at 2.3 Å resoln. Our binary complex structure is the first structure of MurA bound with the substrate UDP- N-acetylglucosamine and thus our study complements the previous structures. The binary and ternary complex structures of H. influenzae MurA are highly similar to each other, both displaying a half-open conformation for the active site loop. This is in sharp contrast with the closed conformation of the corresponding loop in the E. coli MurA ternary complex with UDP-N-acetylglucosamine and fosfomycin. Our results will be useful in structure-based design of specific inhibitors of MurA for antibacterial discovery.
    20. 20
      Minasov, G., Shuvalova, L., Dubrovska, I., Kiryukhina, O., Grimshaw, S., Kwon, K., and Anderson, W. F. (2017) 2.0 Angstrom Resolution Crystal Structure of UDP-N-acetylglucosamine 1-carboxyvinyltransferase from Streptococcus pneumoniae in Complex with Uridine-diphosphate-2(n-acetylglucosaminyl) butyric acid, (2R)-2-(phosphonooxy)propanoic acid and Magnesium. Worldwide Protein Data Bank 5WI5,  DOI: 10.2210/pdb5WI5/pdb
      There is no corresponding record for this reference.
    21. 21
      Schurter, B. T. and Aswad, D. W. (2000) Analysis of isoaspartate in peptides and proteins without the use of radioisotopes. Anal. Biochem. 282, 227– 231,  DOI: 10.1006/abio.2000.4601
      21
      Analysis of Isoaspartate in Peptides and Proteins without the Use of Radioisotopes
      Schurter, Brandon T.; Aswad, Dana W.
      Analytical Biochemistry (2000), 282 (2), 227-231CODEN: ANBCA2; ISSN:0003-2697. (Academic Press)
      A rapid and sensitive HPLC-based method for quantitating isoaspartate levels in peptides and proteins is described. The analyte is incubated for 40 min with S-adenosyl-l-methionine and the com. available enzyme protein L-isoaspartyl methyltransferase. Methylation of isoaspartyl sites results in stoichiometric prodn. of S-adenosyl-L-homocysteine that is sepd. from the other components of the reaction by reversed-phase HPLC and quantitated online by absorbance at 260 nm. This method can accurately detect 5 pmol or less of isoaspartate and works with tryptic digests as well as intact proteins. Using a com. available isoaspartyl peptide, the relationship between isoaspartate levels and S-adenosyl-l-homocysteine prodn. was found to be linear and stoichiometric over a range of 5-250 pmol. Compared to methods that measure [3H]methanol prodn. after methylation with S-adenosyl-l-[methyl-3H]methionine, the HPLC method is safer, faster, less expensive, and equally sensitive. (c) 2000 Academic Press.
    22. 22
      Masson, G. R., Burke, J. E., Ahn, N. G., Anand, G. S., Borchers, C., Brier, S., Bou-Assaf, G. M., Engen, J. R., Englander, S. W., Faber, J., Garlish, R., Griffin, P. R., Gross, M. L., Guttman, M., Hamuro, Y., Heck, A. J. R., Houde, D., Iacob, R. E., Jorgensen, T. J. D., Kaltashov, I. A., Klinman, J. P., Konermann, L., Man, P., Mayne, L., Pascal, B. D., Reichmann, D., Skehel, M., Snijder, J., Strutzenberg, T. S., Underbakke, E. S., Wagner, C., Wales, T. E., Walters, B. T., Weis, D. D., Wilson, D. J., Wintrode, P. L., Zhang, Z., Zheng, J., Schriemer, D. C., and Rand, K. D. (2019) Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments. Nat. Methods 16, 595– 602,  DOI: 10.1038/s41592-019-0459-y
      22
      Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments
      Masson, Glenn R.; Burke, John E.; Ahn, Natalie G.; Anand, Ganesh S.; Borchers, Christoph; Brier, Sebastien; Bou-Assaf, George M.; Engen, John R.; Englander, S. Walter; Faber, Johan; Garlish, Rachel; Griffin, Patrick R.; Gross, Michael L.; Guttman, Miklos; Hamuro, Yoshitomo; Heck, Albert J. R.; Houde, Damian; Iacob, Roxana E.; Joergensen, Thomas J. D.; Kaltashov, Igor A.; Klinman, Judith P.; Konermann, Lars; Man, Petr; Mayne, Leland; Pascal, Bruce D.; Reichmann, Dana; Skehel, Mark; Snijder, Joost; Strutzenberg, Timothy S.; Underbakke, Eric S.; Wagner, Cornelia; Wales, Thomas E.; Walters, Benjamin T.; Weis, David D.; Wilson, Derek J.; Wintrode, Patrick L.; Zhang, Zhongqi; Zheng, Jie; Schriemer, David C.; Rand, Kasper D.
      Nature Methods (2019), 16 (7), 595-602CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophys. technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, anal., presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further addns. and refinements as the field advances.
    23. 23
      Lau, A. M. C., Ahdash, Z., Martens, C., and Politis, A. (2019) Deuteros: software for rapid analysis and visualization of data from differential hydrogen deuterium exchange-mass spectrometry. Bioinformatics 35, 3171– 3173,  DOI: 10.1093/bioinformatics/btz022
      23
      Deuteros: software for rapid analysis and visualization of data from differential hydrogen deuterium exchange-mass spectrometry
      Lau, Andy M. C.; Ahdash, Zainab; Martens, Chloe; Politis, Argyris
      Bioinformatics (2019), 35 (17), 3171-3173CODEN: BOINFP; ISSN:1367-4811. (Oxford University Press)
      Hydrogen deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful technique for interrogating the conformational dynamics of proteins and their complexes. Currently, anal. of HDX-MS data remains a laborious procedure, mainly due to the lack of streamlined software to process the large datasets. We present Deuteros which is a standalone software designed to be coupled with Waters DynamX HDX data anal. software, allowing the rapid anal. and visualization of data from differential HDX-MS.
    24. 24
      Brown, E. D., Vivas, E. I., Walsh, C. T., and Kolter, R. (1995) MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli. J. Bacteriol. 177, 4194– 4197,  DOI: 10.1128/JB.177.14.4194-4197.1995
      24
      MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli
      Brown, Eric D.; Vivas, Eugenio I.; Walsh, Christopher T.; Kolter, Roberto
      Journal of Bacteriology (1995), 177 (14), 4194-7CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      The E. coli gene murZ was recently shown to encode UDP-N-acetylglucosamine enolpyruvyl transferase, which catalyzes the 1st committed step of peptidoglycan biosynthesis. The map position of murZ (69.3 min) differed from that detd. for murA (90 min), a gene which had been previously proposed to encode the same activity. Here, the construction of a chromosomal deletion of murZ and a plasmid contg. murZ under arabinose control is described. Growth of cells contg. the murZ deletion was dependent on the expression of murZ from the plasmid. MurZ apparently is an essential gene and encodes the sole UDP-N-acetylglucosamine enolpyruvyl transferase of E. coli. To simplify the nomenclature, it is recommended that murA be used to designate the gene at 69.3 min that encodes this activity and that the designation murZ be abandoned.
    25. 25
      Johnson, B. A. and Aswad, D. W. (1991) Optimal conditions for the use of protein L-isoaspartyl methyltransferase in assessing the isoaspartate content of peptides and proteins. Anal. Biochem. 192, 384– 391,  DOI: 10.1016/0003-2697(91)90553-6
      25
      Optimal conditions for the use of protein L-isoaspartyl methyltransferase in assessing the isoaspartate content of peptides and proteins
      Johnson, Brett A.; Asward, Dana W.
      Analytical Biochemistry (1991), 192 (2), 384-91CODEN: ANBCA2; ISSN:0003-2697.
      Protein L-isoaspartyl methyltransferase provides a basis for enzymic measurement of atypical, isoaspartyl linkages which make a major contribution to protein microheterogeneity. The low Vmax of the methyltransferase reaction and the instability of the Me ester can hinder accurate detns., and different labs. using different conditions have achieved discrepant values for the isoaspartate content of the same proteins. To investigate the effects of these conditions, and to optimize the assay, isoaspartyl δ sleep-inducing peptide was methylated under a variety of conditions. One μM methyltransferase was required to obtain stoichiometric modification of 2 μM peptide in 40-min reactions at pH 6.2 and 30°. A computer model utilizing kinetic consts. obtained from studies on initial rates of methylation predicted the same requirement for enzyme concn. Carrier protein was necessary for optimal methyltransferase activity at enzyme concns. below 0.4 μM. Stoichiometric methylation required concns. of S-adenosylmethionine to be in substantial excess over those of peptide; 50 μM S-adenosylmethionine is the min. needed for complete modification of 10 μM peptide. Spontaneous demethylation was significant under all conditions tested, so that the Me ester itself never reached a ratio of 1 mol/mol of total peptide. These results demonstrate that the most accurate measurements of isoaspartate are obtained when reactions are carried out at low peptide concns., high S-adenosylmethionine concns., and high enzyme concns. Moreover, quantitation will be significantly better when based on measurements of both methanol and Me esters rather than on measurements of intact Me esters alone.
    26. 26
      Griffith, S. C., Sawaya, M. R., Boutz, D. R., Thapar, N., Katz, J. E., Clarke, S., and Yeates, T. O. (2001) Crystal structure of a protein repair methyltransferase from Pyrococcus furiosus with its L-isoaspartyl peptide substrate. J. Mol. Biol. 313, 1103– 1116,  DOI: 10.1006/jmbi.2001.5095
      26
      Crystal Structure of a Protein Repair Methyltransferase from Pyrococcus furiosus with its L-Isoaspartyl Peptide Substrate
      Griffith, Scott C.; Sawaya, Michael R.; Boutz, Daniel R.; Thapar, Nitika; Katz, Jonathan E.; Clarke, Steven; Yeates, Todd O.
      Journal of Molecular Biology (2001), 313 (5), 1103-1116CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
      Protein L-isoaspartyl (D-aspartyl) methyltransferases (E.C. 2.1.1.77) are found in almost all organisms. These enzymes catalyze the S-adenosylmethionine (AdoMet)-dependent methylation of isomerized and racemized aspartyl residues in age-damaged proteins as part of an essential protein repair process. Here, we report crystal structures of the repair methyltransferase at resolns. up to 1.2 Å from the hyperthermophilic archaeon Pyrococcus furiosus. Refined structures include binary complexes with the active cofactor AdoMet, its reaction product S-adenosylhomocysteine (AdoHcy), and adenosine. The enzyme places the methyl-donating cofactor in a deep, electrostatically neg. pocket that is shielded from solvent. Across the multiple crystal structures visualized, the presence or absence of the Me group on the cofactor correlates with a significant conformational change in the enzyme in a loop bordering the active site, suggesting a role for motion in catalysis or cofactor exchange. We also report the structure of a ternary complex of the enzyme with adenosine and the methyl-accepting polypeptide substrate VYP(L-isoAsp)HA at 2.1 Å. The substrate binds in a narrow active site cleft with three of its residues in an extended conformation, suggesting that damaged proteins may be locally denatured during the repair process in cells. Manual and computer-based docking studies on different isomers help explain how the enzyme uses steric effects to make the crit. distinction between normal L-aspartyl and age-damaged L-isoaspartyl and D-aspartyl residues. (c) 2001 Academic Press.
    27. 27
      Bensen, D. C., Rodriguez, S., Nix, J., Cunningham, M. L., and Tari, L. W. (2012) Structure of MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) from Vibrio fischeri in complex with substrate UDP-N-acetylglucosamine and the drug fosfomycin. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 68, 382– 385,  DOI: 10.1107/S1744309112006720
      27
      Structure of MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) from Vibrio fischeri in complex with substrate UDP-N-acetylglucosamine and the drug fosfomycin
      Bensen, D. C.; Rodriguez, S.; Nix, J.; Cunningham, M. L.; Tari, L. W.
      Acta Crystallographica, Section F: Structural Biology and Crystallization Communications (2012), 68 (4), 382-385CODEN: ACSFCL; ISSN:1744-3091. (International Union of Crystallography)
      The development of new antibiotics is necessitated by the rapid development of resistance to current therapies. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), which catalyzes the first committed step of bacterial peptidoglycan biosynthesis, is a prime candidate for therapeutic intervention. MurA is the target of the antibiotic fosfomycin, a natural product produced by Streptomyces. Despite possessing a high degree of sequence conservation with MurA enzymes from fosfomycin-susceptible organisms, recent microbiol. studies suggest that MurA from Vibrio fischeri (VfiMurA) may confer fosfomycin resistance via a mechanism that is not yet understood. The crystal structure of VfiMurA in a ternary complex with the substrate UDP-N-acetylglucosamine (UNAG) and fosfomycin has been solved to a resoln. of 1.93 Å. Fosfomycin is known to inhibit MurA by covalently binding to a highly conserved cysteine in the active site of the enzyme. A comparison of the title structure with the structure of fosfomycin-susceptible Haemophilus influenzae MurA (PDB entry ) revealed strikingly similar conformations of the mobile substrate-binding loop and clear electron d. for a fosfomycin-cysteine adduct. Based on these results, there are no distinguishing sequence/structural features in VfiMurA that would translate to a diminished sensitivity to fosfomycin. However, VfiMurA is a robust crystallizer and shares high sequence identity with many clin. relevant bacterial pathogens. Thus, it would serve as an ideal system for use in the structure-guided optimization of new antibacterial agents.
    28. 28
      Robinson, N. E. (2002) Protein deamidation. Proc. Natl. Acad. Sci. U. S. A. 99, 5283– 5288,  DOI: 10.1073/pnas.082102799
      28
      Protein deamidation
      Robinson, Noah E.
      Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (8), 5283-5288CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      A completely automatic computerized technique for the quant. estn. of the deamidation rates of any protein for which the three-dimensional structure is known has been developed. Calcns. of the specific deamidation rates of 170,014 asparaginyl residues in 13,335 proteins have been carried out. The calcd. values have good quant. reliability when compared with exptl. measurements. These rates demonstrate that deamidation may be a biol. relevant phenomenon in a remarkably large percentage of proteins.
    29. 29
      Dai, S., Lee, D. H., Kenton, N. T., and Zhou, Z. S. (2013) Peptidyl-Asp Metalloendopeptidase. In Handbook of Proteolytic Enzymes 1281– 1285,  DOI: 10.1016/B978-0-12-382219-2.00288-X
      There is no corresponding record for this reference.
    30. 30
      Lanzetta, P. A., Alvarez, L. J., Reinach, P. S., and Candia, O. A. (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem. 100, 95– 97,  DOI: 10.1016/0003-2697(79)90115-5
      30
      An improved assay for nanomole amounts of inorganic phosphate
      Lanzetta, Peter A.; Alvarez, Lawrence J.; Reinach, Peter S.; Candia, Oscar A.
      Analytical Biochemistry (1979), 100 (1), 95-7CODEN: ANBCA2; ISSN:0003-2697.
      A colorimetric assay for the detn. of nanomol amts. of inorg. phosphate is described. The procedure combines a very high molar absorptivity with color stability and insensitivity to newly released phosphate from labile organophosphates. The quenching procedure of E. E. Baginski et al. (1975) was modified for use with the method of H. H. Hess and J. E. Derr (1975). The color reagent was a mixt. of 0.045% Malachite green-HCl and 4.2% NH4 molybdate in 4N HCl + Sterox. Solns. were read at 660 nm.
    31. 31
      Clarke, J. and Fersht, A. R. (1993) Engineered disulfide bonds as probes of the folding pathway of barnase: increasing the stability of proteins against the rate of denaturation. Biochemistry 32, 4322– 4329,  DOI: 10.1021/bi00067a022
      31
      Engineered disulfide bonds as probes of the folding pathway of barnase: Increasing the stability of proteins against the rate of denaturation
      Clarke, Jane; Fersht, Alan R.
      Biochemistry (1993), 32 (16), 4322-9CODEN: BICHAW; ISSN:0006-2960.
      Disulfide bridges have been introduced into barnase to act as probes of folding. One disulfide (between residues 85 and 102) links two loops known to pack together early in the folding pathway. A second disulfide bond (between residues 43 and 80) links two elements of secondary structure known to pack together only after the rate-detg. step of folding. The disulfide-bridged proteins are more stable than wild-type by 4.1 and 1.2 kcal mol-1, resp. The kinetics of unfolding and refolding of the mutant proteins has been measured, and a comparison of the disulfide proteins and their corresponding dithiol forms has been made by use of thermodn. cycles. These data have been used to construct folding profiles of the disulfide proteins. The disulfide bond engineered into the part of the protein that folds early confers stability upon the intermediate and transition states of folding. The protein with a disulfide bond connecting parts of the protein that fold late is not stabilized until the protein reaches its final folded form. Conversely, in the unfolding pathway, the rate of unfolding of this mutant is lowered considerably. This points to a method of decreasing the rate of denaturation of proteins that are used in medical and biotechnol. applications: elements of structure that unfold in or before the rate-detg. step of overall unfolding may be stabilized and so slow down the overall unfolding process. The barnase mutant linked between Cys 43 and Cys 80, for example, unfolds 20 times slower than wild-type and 170 times slower than the reduced protein.
    32. 32
      Fersht, A. (1999) Structure And Mechanism in Protein Science, W. H. Freeman and Company.
      There is no corresponding record for this reference.
    33. 33
      Engen, J. R. (2009) Analysis of protein conformation and dynamics by hydrogen/deuterium exchange MS. Anal. Chem. 81, 7870– 7875,  DOI: 10.1021/ac901154s
      33
      Analysis of Protein Conformation and Dynamics by Hydrogen/Deuterium Exchange MS
      Engen, John R.
      Analytical Chemistry (Washington, DC, United States) (2009), 81 (19), 7870-7875CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      A review. Understanding as much as possible about proteins in the shortest amt. of time has long been a goal of hydrogen exchange MS [mass spectrometry]. Recent technol. advances in hydrogen exchange MS have led to improvements in the technique, but has this goal yet been achieved.
    34. 34
      Konermann, L., Pan, J., and Liu, Y. H. (2011) Hydrogen exchange mass spectrometry for studying protein structure and dynamics. Chem. Soc. Rev. 40, 1224– 1234,  DOI: 10.1039/C0CS00113A
      34
      Hydrogen exchange mass spectrometry for studying protein structure and dynamics
      Konermann, Lars; Pan, Jingxi; Liu, Yu-Hong
      Chemical Society Reviews (2011), 40 (3), 1224-1234CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has become a key technique for monitoring structural and dynamic aspects of proteins in soln. This approach relies on the fact that exposure of a protein to D2O induces rapid amide H D exchange in disordered regions that lack stable hydrogen-bonding. Tightly folded elements are much more protected from HDX, resulting in slow isotope exchange that is mediated by the structural dynamics ("breathing motions") of the protein. MS-based peptide mapping is a well established technique for measuring the mass shifts of individual protein segments. This tutorial review briefly discusses basic fundamentals of HDX/MS, before highlighting a no. of recent developments and applications. Gas phase fragmentation strategies represent a promising alternative to the traditional proteolysis-based approach, but experimentalists have to be aware of scrambling phenomena that can be encountered under certain conditions. Electron-based dissocn. methods provide a soln. to this problem. We also discuss recent advances that facilitate the applicability of HDX/MS to membrane proteins, and to the characterization of short-lived protein folding intermediates. It is hoped that this review will provide a starting point for novices, as well as a useful ref. for practitioners, who require an overview of some recent trends in HDX/MS.
    35. 35
      Martens, C., Shekhar, M., Borysik, A. J., Lau, A. M., Reading, E., Tajkhorshid, E., Booth, P. J., and Politis, A. (2018) Direct protein-lipid interactions shape the conformational landscape of secondary transporters. Nat. Commun. 9, 4151,  DOI: 10.1038/s41467-018-06704-1
      35
      Direct protein-lipid interactions shape the conformational landscape of secondary transporters
      Martens Chloe; Borysik Antoni J; Lau Andy M; Reading Eamonn; Booth Paula J; Politis Argyris; Shekhar Mrinal; Tajkhorshid Emad
      Nature communications (2018), 9 (1), 4151 ISSN:.
      Secondary transporters undergo structural rearrangements to catalyze substrate translocation across the cell membrane - yet how such conformational changes happen within a lipid environment remains poorly understood. Here, we combine hydrogen-deuterium exchange mass spectrometry (HDX-MS) with molecular dynamics (MD) simulations to understand how lipids regulate the conformational dynamics of secondary transporters at the molecular level. Using the homologous transporters XylE, LacY and GlpT from Escherichia coli as model systems, we discover that conserved networks of charged residues act as molecular switches that drive the conformational transition between different states. We reveal that these molecular switches are regulated by interactions with surrounding phospholipids and show that phosphatidylethanolamine interferes with the formation of the conserved networks and favors an inward-facing state. Overall, this work provides insights into the importance of lipids in shaping the conformational landscape of an important class of transporters.
    36. 36
      Müller, M. M. (2018) Post-Translational Modifications of Protein Backbones: Unique Functions, Mechanisms, and Challenges. Biochemistry 57, 177– 185,  DOI: 10.1021/acs.biochem.7b00861
      36
      Post-Translational Modifications of Protein Backbones: Unique Functions, Mechanisms, and Challenges
      Muller, Manuel M.
      Biochemistry (2018), 57 (2), 177-185CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      A review. Post-translational modifications (PTMs) dramatically enhance the capabilities of proteins. They introduce new functionalities and dynamically control protein activity by modulating intra- and intermol. interactions. Traditionally, PTMs have been considered as reversible attachments to nucleophilic functional groups on amino acid side chains, whereas the polypeptide backbone is often thought to be inert. This paradigm is shifting as chem. and functionally diverse alterations of the protein backbone are discovered. Importantly, backbone PTMs can control protein structure and function just as side chain modifications do, and operate through unique mechanisms to achieve these features. In this perspective, I outline the various types of protein backbone modifications discovered so far and highlight their contributions to biol. as well as the challenges faced in studying these versatile PTMs.
    37. 37
      Mamula, M. J., Gee, R. J., Elliott, J. I., Sette, A., Southwood, S., Jones, P.-J., and Blier, P. R. (1999) Isoaspartyl Post-translational Modification Triggers Autoimmune Responses to Self-proteins. J. Biol. Chem. 274, 22321– 22327,  DOI: 10.1074/jbc.274.32.22321
      37
      Isoaspartyl post-translational modification triggers autoimmune responses to self-proteins
      Mamula, Mark J.; Gee, Renelle J.; Elliott, James I.; Sette, Alessandro; Southwood, Scott; Jones, Paul-James; Blier, Peter R.
      Journal of Biological Chemistry (1999), 274 (32), 22321-22327CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The normal functioning immune system is programmed to attack foreign pathogens and other foreign proteins while maintaining tolerance to self-proteins. The mechanisms by which tolerance is broken in the initiation of autoimmunity are not completely understood. In the present study, mice immunized with the murine cytochrome c peptide 90-104 showed no response by the B or T cell compartments. However, immunization with the isoaspartyl form of this peptide, where the linkage of Asp93 to Leu94 occurs through the β-carboxyl group, resulted in strong B and T cell autoimmune responses. Antibodies elicited by immunization with the isoaspartyl form of self-peptide were cross-reactive in binding to both isoforms of cytochrome c peptide and to native cytochrome c self-protein. In a similar manner, immunization of mice with the isoaspartyl form of a peptide autoantigen of human systemic lupus erythematosus (SLE) resulted in strong B and T cell responses while mice maintained tolerance to the normal aspartyl form of self-antigen. Isoaspartyl linkages within proteins are enhanced in aging and stressed cells and arise under physiol. conditions. These post-translationally modified peptides may serve as an early immunol. stimulus in autoimmune disease.
    38. 38
      Manning, M. C., Chou, D. K., Murphy, B. M., Payne, R. W., and Katayama, D. S. (2010) Stability of Protein Pharmaceuticals: An Update. Pharm. Res. 27, 544– 575,  DOI: 10.1007/s11095-009-0045-6
      38
      Stability of protein pharmaceuticals: an update
      Manning Mark Cornell; Chou Danny K; Murphy Brian M; Payne Robert W; Katayama Derrick S
      Pharmaceutical research (2010), 27 (4), 544-75 ISSN:.
      In 1989, Manning, Patel, and Borchardt wrote a review of protein stability (Manning et al., Pharm. Res. 6:903-918, 1989), which has been widely referenced ever since. At the time, recombinant protein therapy was still in its infancy. This review summarizes the advances that have been made since then regarding protein stabilization and formulation. In addition to a discussion of the current understanding of chemical and physical instability, sections are included on stabilization in aqueous solution and the dried state, the use of chemical modification and mutagenesis to improve stability, and the interrelationship between chemical and physical instability.
    39. 39
      Yokoyama, H., Mizutani, R., Noguchi, S., and Hayashida, N. (2019) Structural and biochemical basis of the formation of isoaspartate in the complementarity-determining region of antibody 64M-5 Fab. Sci. Rep. 9, 9,  DOI: 10.1038/s41598-019-54918-0
      There is no corresponding record for this reference.
    40. 40
      Jefferis, R. (2016) Posttranslational Modifications and the Immunogenicity of Biotherapeutics. J. Immunol. Res. 2016, 5358272,  DOI: 10.1155/2016/5358272
      40
      Posttranslational Modifications and the Immunogenicity of Biotherapeutics
      Jefferis Roy
      Journal of immunology research (2016), 2016 (), 5358272 ISSN:.
      Whilst the amino acid sequence of a protein is determined by its gene sequence, the final structure and function are determined by posttranslational modifications (PTMs), including quality control (QC) in the endoplasmic reticulum (ER) and during passage through the Golgi apparatus. These processes are species and cell specific and challenge the biopharmaceutical industry when developing a production platform for the generation of recombinant biologic therapeutics. Proteins and glycoproteins are also subject to chemical modifications (CMs) both in vivo and in vitro. The individual is naturally tolerant to molecular forms of self-molecules but nonself variants can provoke an immune response with the generation of anti-drug antibodies (ADA); aggregated forms can exhibit enhanced immunogenicity and QC procedures are developed to avoid or remove them. Monoclonal antibody therapeutics (mAbs) are a special case because their purpose is to bind the target, with the formation of immune complexes (ICs), a particular form of aggregate. Such ICs may be removed by phagocytic cells that have antigen presenting capacity. These considerations may frustrate the possibility of ameliorating the immunogenicity of mAbs by rigorous exclusion of aggregates from drug product. Alternate strategies for inducing immunosuppression or tolerance are discussed.
    41. 41
      de Brevern, A. G. (2016) Extension of the classical classification of β-turns. Sci. Rep. 6, 33191,  DOI: 10.1038/srep33191
      41
      Extension of the classical classification of β-turns
      de Brevern, Alexandre G.
      Scientific Reports (2016), 6 (), 33191CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      The functional properties of a protein primarily depend on its three-dimensional (3D) structure. These properties have classically been assigned, visualized and analyzed on the basis of protein secondary structures. The β-turn is the third most important secondary structure after helixes and β-strands. β-Turns have been classified according to the values of the dihedral angles φ and ψ of the central residue. Conventionally, eight different types of β-turns have been defined, whereas those that cannot be defined are classified as type IV β-turns. This classification remains the most widely used. Nonetheless, the misc. type IV β-turns represent 1/3rd of β-turn residues. An unsupervised specific clustering approach was designed to search for recurrent new turns in the type IV category. The classical rules of β-turn type assignment were central to the approach. The four most frequently occurring clusters defined the new β-turn types. Unexpectedly, these types, designated IV1, IV2, IV3 and IV4, represent half of the type IV β-turns and occur more frequently than many of the previously established types. These types show convincing particularities, in terms of both structures and sequences that allow for the classical β-turn classification to be extended for the first time in 25 years.
    42. 42
      Chatterjee, S., Roy, R. S., and Balaram, P. (2007) Expanding the polypeptide backbone: hydrogen-bonded conformations in hybrid polypeptides containing the higher homologues of alpha-amino acids. J. R. Soc., Interface 4, 587– 606,  DOI: 10.1098/rsif.2006.0203
      42
      Expanding the polypeptide backbone: hydrogen-bonded conformations in hybrid polypeptides containing the higher homologues of α-amino acids
      Chatterjee, Sunanda; Roy, Rituparna Sinha; Balaram, P.
      Journal of the Royal Society, Interface (2007), 4 (15), 587-606CODEN: JRSICU; ISSN:1742-5689. (Royal Society)
      A review. Half a century has passed since the hydrogen-bonded secondary structures of polypeptides and proteins were first recognized. An extraordinary wealth of conformational information is now available on peptides and proteins, which are formed of α-amino acid residues. More recently, the discovery of well-folded structures in oligopeptides contg. β-amino acids has focused a great deal of current interest on the conformational properties of peptides constructed from higher homologues (ω) of α-amino acids. This review examines the nature of intramolecularly hydrogen-bonded conformations of hybrid peptides formed by amino acid residues, with a varying no. of backbone atoms. The β-turn, a ubiquitous structural feature formed by two residue (αα) segments in proteins and peptides, is stabilized by a 10-atom (C10) intramol. 4→1 hydrogen bond. Hybrid turns may be classified by comparison with their αα counterparts. The available crystallog. information on hydrogen-bonded hybrid turns is surveyed in this review. Several recent examples demonstrate that individual ω-amino acid residues and hybrid dipeptide segments may be incorporated into the regular structures of α-peptides. Examples of both peptide helixes and hairpins are presented. The present review explores the relationships between folded conformations in hybrid sequences and their counterparts in all α-residue sequences. The use of stereochem. constrained ω-residues promises to expand the range of peptide design strategies to include ω-amino acids. This approach is exemplified by well-folded structures like the C12 (αγ) and C14 (γγ) helixes formed in short peptides contg. multiply substituted γ-residues. The achiral γ-residue gabapentin is a readily accessible building block in the design of peptides contg. γ-amino acids. The construction of globular polypeptide structures using diverse hybrid sequences appears to be a realistic possibility.
    43. 43
      George, K. L. and Horne, W. S. (2018) Foldamer Tertiary Structure through Sequence-Guided Protein Backbone Alteration. Acc. Chem. Res. 51, 1220– 1228,  DOI: 10.1021/acs.accounts.8b00048
      43
      Foldamer Tertiary Structure through Sequence-Guided Protein Backbone Alteration
      George, Kelly L.; Horne, W. Seth
      Accounts of Chemical Research (2018), 51 (5), 1220-1228CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. The prospect of recreating the complex structural hierarchy of protein folding in synthetic oligomers with backbones that are artificial in covalent structure ("foldamers") has long fascinated chemists. Foldamers offer complex functions from biostable scaffolds and have found widespread applications in fields from biomedical to materials science. Most precedent has focused on isolated secondary structures or their assemblies. In considering the goal of complex protein-like tertiary folding patterns, a key barrier became apparent. How does one design a backbone with covalent connectivity and a sequence of side-chain functional groups that will support defined intramol. packing of multiple artificial secondary structures. Two developments were key to overcoming this challenge. First was the recognition of the power of blending α-amino acid residues with monomers differing in backbone connectivity to create "heterogeneous-backbone" foldamers. Second was the finding that replacing some of the natural α-residues in a biol. sequence with artificial-backbone variants can result in a mimic that retains both the fold and function of the native sequence and, in some cases, gains advantageous characteristics. Taken together, these precedents lead to a view of a protein as chem. entity having two orthogonal sequences: a sequence of side-chain functional groups and a sep. sequence of backbone units displaying those functional groups. In this Account, the authors describe the lab's work over the last ∼10 years to leverage the above concept of protein sequence duality in order to develop design principles for constructing heterogeneous-backbone foldamers that adopt complex protein-like tertiary folds. Fundamental to the approach is the utilization of a variety of artificial building blocks (e.g., D-α-residues, Cα-Me-α-residues, N-Me-α-residues, β-residues, γ-residues, δ-residues, polymer segments) in concert, replacing a fraction of α-residues in a given prototype sequence. The authors provide an overview of the state-of-the-art in terms of design principles for choosing substitutions based on consideration of local secondary structure and retention of key side-chain functional groups. The authors survey high-resoln. structures of backbone-modified proteins to illustrate how diverse artificial moieties are accommodated in tertiary fold contexts. The authors detail efforts to elucidate how backbone alteration impacts folding thermodn. and describe how such data informs the development of improved design rules. Collectively, a growing body of results by the lab and others spanning multiple protein systems suggests there is a great deal of plasticity with respect to the backbone chem. structures upon which sequence-encoded tertiary folds can manifest. Moreover, these efforts suggest sequence-guided backbone alteration as a broadly applicable strategy for generating foldamers with complex tertiary folding patterns. The authors conclude by offering some perspective regarding the near future of this field, in terms of unanswered questions, technol. needs, and opportunities for new areas of inquiry.
    44. 44
      Kreitler, D. F., Mortenson, D. E., Forest, K. T., and Gellman, S. H. (2016) Effects of Single alpha-to-beta Residue Replacements on Structure and Stability in a Small Protein: Insights from Quasiracemic Crystallization. J. Am. Chem. Soc. 138, 6498– 6505,  DOI: 10.1021/jacs.6b01454
      44
      Effects of Single α-to-β Residue Replacements on Structure and Stability in a Small Protein: Insights from Quasiracemic Crystallization
      Kreitler, Dale F.; Mortenson, David E.; Forest, Katrina T.; Gellman, Samuel H.
      Journal of the American Chemical Society (2016), 138 (20), 6498-6505CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Synthetic peptides that contain backbone modifications but nevertheless adopt folded structures similar to those of natural polypeptides are of fundamental interest and may provide a basis for biomedical applications. Such mols. can, for example, mimic the ability of natural prototypes to bind to specific target macromols. but resist degrdn. by proteases. We have previously shown that oligomers contg. mixts. of α- and β-amino acid residues ("α/β-peptides") can mimic the α-helix secondary structure, and that properly designed α/β-peptides can bind to proteins that evolved to bind to α-helical partners. Here we report fundamental studies that support the long-range goal of extending the α/β approach to tertiary structures. We have evaluated the impact of single α → β modifications on the structure and stability of the small and well-studied villin headpiece subdomain (VHP). The native state of this 35-residue polypeptide contains several α-helical segments packed around a small hydrophobic core. We examd. α → β substitution at four solvent-exposed positions, Asn19, Trp23, Gln26 and Lys30. In each case, both the β3 homolog of the natural α residue and a cyclic β residue were evaluated. All α → β3 substitutions caused significant destabilization of the tertiary structure as measured by variable-temp. CD, although at some of these positions, replacing the β3 residue with a cyclic β residue led to improved stability. Atomic-resoln. structures of four VHP analogs were obtained via quasiracemic crystn. These findings contribute to a fundamental α/β-peptide knowledge-base by confirming that β3-amino acid residues can serve as effective structural mimics of homologous α-amino acid residues within a natural tertiary fold, which should support rational design of functional α/β analogs of natural poly-α-peptides.
    45. 45
      Reinert, Z. E., Lengyel, G. A., and Horne, W. S. (2013) Protein-like tertiary folding behavior from heterogeneous backbones. J. Am. Chem. Soc. 135, 12528– 12531,  DOI: 10.1021/ja405422v
      45
      Protein-like Tertiary Folding Behavior from Heterogeneous Backbones
      Reinert, Zachary E.; Lengyel, George A.; Horne, W. Seth
      Journal of the American Chemical Society (2013), 135 (34), 12528-12531CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Because proteins play vital roles in life, much effort has been invested in their mimicry by synthetic agents. One approach is to design unnatural backbone oligomers (foldamers) that fold like natural peptides. Despite success in secondary structure mimicry by such species, protein-like tertiary folds remain elusive. A fundamental challenge underlying this task is the design of a sequence of side chains that will specify a complex tertiary folding pattern on an unnatural backbone. We report here a sequence-based approach to convert a natural protein with a compact tertiary fold to an analog with a backbone composed of ∼20% unnatural building blocks but folding behavior similar to that of the parent protein.
    46. 46
      Horne, W. S., Price, J. L., and Gellman, S. H. (2008) Interplay among side chain sequence, backbone composition, and residue rigidification in polypeptide folding and assembly. Proc. Natl. Acad. Sci. U. S. A. 105, 9151– 9156,  DOI: 10.1073/pnas.0801135105
      46
      Interplay among side chain sequence, backbone composition, and residue rigidification in polypeptide folding and assembly
      Horne, W. Seth; Price, Joshua L.; Gellman, Samuel H.
      Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (27), 9151-9156CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The extent to which polypeptide conformation depends on side-chain compn. and sequence has been widely studied, but less is known about the importance of maintaining an α-amino acid backbone. Here, we examine a series of peptides with backbones that feature different repeating patterns of α- and β-amino acid residues but an invariant side-chain sequence. In the pure α-backbone, this sequence corresponds to the previously studied peptide GCN4-pLI, which forms a very stable four-helix bundle quaternary structure. Phys. characterization in soln. and crystallog. structure detn. show that a variety of α/β-peptide backbones can adopt sequence-encoded quaternary structures similar to that of the a prototype. There is a loss in helix bundle stability upon β-residue incorporation; however, stability of the quaternary structure is not a simple function of β-residue content. We find that cyclically constrained β-amino acid residues can stabilize the folds of α/β-peptide GCN4-pLI analogs and restore quaternary structure formation to backbones that are predominantly unfolded in the absence of cyclic residues. Our results show a surprising degree of plasticity in terms of the backbone compns. that can manifest the structural information encoded in a sequence of amino acid side chains. These findings offer a framework for the design of nonnatural oligomers that mimic the structural and functional properties of proteins.
    47. 47
      Lengyel, G. A. and Horne, W. S. (2012) Design strategies for the sequence-based mimicry of side-chain display in protein beta-sheets by alpha/beta-peptides. J. Am. Chem. Soc. 134, 15906– 15913,  DOI: 10.1021/ja306311r
      47
      Design Strategies for the Sequence-Based Mimicry of Side-Chain Display in Protein β-Sheets by α/β-Peptides
      Lengyel, George A.; Horne, W. Seth
      Journal of the American Chemical Society (2012), 134 (38), 15906-15913CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The sophistication of folding patterns and functions displayed by unnatural-backbone oligomers has increased tremendously in recent years. Design strategies for the mimicry of tertiary structures seem within reach; however, a general method for the mimicry of sheet segments in the context of a folded protein is an unmet need preventing realization of this goal. Previous work has shown that 1→1 α→β-residue substitutions at cross-strand positions in a hairpin-forming α-peptide sequence can generate an α/β-peptide analog that folds in aq. conditions but with a change in side-chain display relative to the natural sequence; this change would prevent application of single β-residue substitutions in a larger protein. Here, we evaluate four different substitution strategies based on replacement of αα dipeptide segments for the ability to retain both sheet folding encoded by a parent α-peptide sequence as well as nativelike side-chain display in the vicinity of the β-residue insertion point. High-resoln. structure detn. and thermodn. anal. of folding by multidimensional NMR suggest that three of the four designs examd. are applicable to larger proteins.
    48. 48
      Arnold, U., Huck, B. R., Gellman, S. H., and Raines, R. T. (2013) Protein prosthesis: beta-peptides as reverse-turn surrogates. Protein Sci. 22, 274– 279,  DOI: 10.1002/pro.2208
      48
      Protein prosthesis: β-peptides as reverse-turn surrogates
      Arnold, Ulrich; Huck, Bayard R.; Gellman, Samuel H.; Raines, Ronald T.
      Protein Science (2013), 22 (3), 274-279CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
      The introduction of non-natural modules could provide unprecedented control over folding/unfolding behavior, conformational stability, and biol. function of proteins. Success requires the interrogation of candidate modules in natural contexts. Here, expressed protein ligation is used to replace a reverse turn in bovine pancreatic RNase (RNase A) with a synthetic β-dipeptide: β2-homoalanine-β3-homoalanine. This segment is known to adopt an unnatural reverse-turn conformation that contains a 10-membered ring hydrogen bond, but one with a donor-acceptor pattern opposite to that in the 10-membered rings of natural reverse turns. The RNase A variant has intact enzymic activity, but unfolds more quickly and has diminished conformational stability relative to native RNase A. These data indicate that hydrogen-bonding pattern merits careful consideration in the selection of beneficial reverse-turn surrogates.
    49. 49
      Arnold, U., Hinderaker, M. P., Nilsson, B. L., Huck, B. R., Gellman, S. H., and Raines, R. T. (2002) Protein prosthesis: a semisynthetic enzyme with a beta-peptide reverse turn. J. Am. Chem. Soc. 124, 8522– 8523,  DOI: 10.1021/ja026114n
      49
      Protein prosthesis: A semisynthetic enzyme with a β-peptide reverse turn
      Arnold, Ulrich; Hinderaker, Matthew P.; Nilsson, Bradley L.; Huck, Bayard R.; Gellman, Samuel H.; Raines, Ronald T.
      Journal of the American Chemical Society (2002), 124 (29), 8522-8523CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      β-Amino acids are incorporated into an enzyme by using the method of expressed protein ligation. In the resulting semisynthetic enzyme, an R-nipecotic acid-S-nipecotic acid module replaces Asn113 and Pro114 of RNase A. The semisynthetic enzyme not only retains full catalytic activity but also gains conformational stability. Thus, structural elements can be replaced with foldameric equiv. to endow proteins with more desirable properties.
    50. 50
      Daura, X., Gademann, K., Schäfer, H., Jaun, B., Seebach, D., and van Gunsteren, W. F. (2001) The β-Peptide Hairpin in Solution: Conformational Study of a β-Hexapeptide in Methanol by NMR Spectroscopy and MD Simulation. J. Am. Chem. Soc. 123, 2393– 2404,  DOI: 10.1021/ja003689g
      50
      The β-Peptide Hairpin in Solution: Conformational Study of a β-Hexapeptide in Methanol by NMR Spectroscopy and MD Simulation
      Daura, Xavier; Gademann, Karl; Schaefer, Heiko; Jaun, Bernhard; Seebach, Dieter; van Gunsteren, Wilfred F.
      Journal of the American Chemical Society (2001), 123 (10), 2393-2404CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The structural and thermodn. properties of a 6-residue β-peptide I that was designed to form a hairpin conformation have been studied by NMR spectroscopy and mol. dynamics simulation in methanol soln. The predicted hairpin would be characterized by a 10-membered hydrogen-bonded turn involving residues 3 and 4, and two extended antiparallel strands. The interproton distances and backbone torsional dihedral angles derived from the NMR expts. at room temp. are in general terms compatible with the hairpin conformation. Two trajectories of system configurations from 100-ns mol.-dynamics simulations of the peptide in soln. at 298 and 340 K have been analyzed. In both simulations, reversible folding to the hairpin conformation is obsd. Interestingly, there is a significant conformational overlap between the unfolded state of the peptide at each of the temps. As already obsd. in previous studies of peptide folding, the unfolded state is composed of a (relatively) small no. of predominant conformers and in this case lacks any type of secondary-structure element. The trajectories provide an excellent ground for the interpretation of the NMR-derived data in terms of ensemble avs. and distributions as opposed to single-conformation interpretations. From this perspective, a relative population of the hairpin conformation of 20% to 30% would suffice to explain the NMR-derived data. Surprisingly, however, the ensemble of structures from the simulation at 340 K reproduces more accurately the NMR-derived data than the ensemble from the simulation at 298 K, and this point needs further investigation.
    51. 51
      Mayer, C., Muller, M. M., Gellman, S. H., and Hilvert, D. (2014) Building proficient enzymes with foldamer prostheses. Angew. Chem., Int. Ed. 53, 6978– 6981,  DOI: 10.1002/anie.201400945
      51
      Building Proficient Enzymes with Foldamer Prostheses
      Mayer, Clemens; Mueller, Manuel M.; Gellman, Samuel H.; Hilvert, Donald
      Angewandte Chemie, International Edition (2014), 53 (27), 6978-6981CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Foldamers are non-natural oligomers that adopt stable conformations reminiscent of those found in proteins. To evaluate the potential of foldameric subunits for catalysis, semisynthetic enzymes contg. foldamer fragments constructed from α- and β-amino acid residues were designed and characterized. Systematic variation of the α→β substitution pattern and types of β-residue afforded highly proficient hybrid catalysts, thus demonstrating the feasibility of expanding the enzyme-engineering toolkit with non-natural backbones.
    52. 52
      Mortenson, D. E., Kreitler, D. F., Thomas, N. C., Guzei, I. A., Gellman, S. H., and Forest, K. T. (2018) Evaluation of beta-Amino Acid Replacements in Protein Loops: Effects on Conformational Stability and Structure. ChemBioChem 19, 604– 612,  DOI: 10.1002/cbic.201700580
      52
      Evaluation of β-Amino Acid Replacements in Protein Loops: Effects on Conformational Stability and Structure
      Mortenson, David E.; Kreitler, Dale F.; Thomas, Nicole C.; Guzei, Ilia A.; Gellman, Samuel H.; Forest, Katrina T.
      ChemBioChem (2018), 19 (6), 604-612CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      β-Amino acids have a backbone that is expanded by one carbon atom relative to α-amino acids, and β residues have been investigated as subunits in protein-like mols. that adopt discrete and predictable conformations. Two classes of β residue have been widely explored in the context of generating α-helix-like conformations: β3-amino acids, which are homologous to α-amino acids and bear a side chain on the backbone carbon adjacent to nitrogen, and residues constrained by a five-membered ring, such the one derived from trans-2-aminocyclopentanecarboxylic acid (ACPC). Substitution of α residues with their β3 homologues within an α-helix-forming sequence generally causes a decrease in conformational stability. Use of a ring-constrained β residue, however, can offset the destabilizing effect of α→β substitution. Here we extend the study of α→β substitutions, involving both β3 and ACPC residues, to short loops within a small tertiary motif. We start from previously reported variants of the Pin1 WW domain that contain a two-, three-, or four-residue β-hairpin loop, and we evaluate α→β replacements at each loop position for each variant. By referral to the .vphi.,ψ angles of the native structure, one can choose a stereochem. appropriate ACPC residue. Use of such logically chosen ACPC residues enhances conformational stability in several cases. Crystal structures of three β-contg. Pin1 WW domain variants show that a native-like tertiary structure is maintained in each case.
    53. 53
      Tsien, R. Y. (1998) THE GREEN FLUORESCENT PROTEIN. Annu. Rev. Biochem. 67, 509– 544,  DOI: 10.1146/annurev.biochem.67.1.509
      53
      The green fluorescent protein
      Tsien, Roger Y.
      Annual Review of Biochemistry (1998), 67 (), 509-544CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
      A review, with ∼114 refs. In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochem. and cell biol. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resoln. crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiol. indicators, biosensors, and photochem. memories.
    54. 54
      Schwede, T. F., Rétey, J., and Schulz, G. E. (1999) Crystal Structure of Histidine Ammonia-Lyase Revealing a Novel Polypeptide Modification as the Catalytic Electrophile. Biochemistry 38, 5355– 5361,  DOI: 10.1021/bi982929q
      54
      Crystal Structure of Histidine Ammonia-Lyase Revealing a Novel Polypeptide Modification as the Catalytic Electrophile
      Schwede, Torsten F.; Retey, Janos; Schulz, Georg E.
      Biochemistry (1999), 38 (17), 5355-5361CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Histidine ammonia-lyase (EC 4.3.1.3) catalyzes the nonoxidative elimination of the α-amino group of histidine and is closely related to the important plant enzyme phenylalanine ammonia-lyase. The crystal structure of histidase from Pseudomonas putida was detd. at 2.1 Å resoln. revealing a homotetramer with D2 symmetry, the mol. center of which is formed by 20 nearly parallel α-helixes. The chain fold, but not the sequence, resembles those of fumarase C and related proteins. The structure shows that the reactive electrophile is a 4-methylidene-imidazole-5-one, which is formed autocatalytically by cyclization and dehydration of residues 142-144 with the sequence Ala-Ser-Gly. With respect to the first dehydration step, this modification resembles the chromophore of the green fluorescent protein. The active center is clearly established by the modification and by mutations. The obsd. geometry allowed us to model the bound substrate at a high confidence level. A reaction mechanism is proposed.
    55. 55
      Kumar, S., Prakash, S., Gupta, K., Dongre, A., Balaram, P., and Balaram, H. (2016) Unexpected functional implication of a stable succinimide in the structural stability of Methanocaldococcus jannaschii glutaminase. Nat. Commun. 7, 12798,  DOI: 10.1038/ncomms12798
      55
      Unexpected functional implication of a stable succinimide in the structural stability of Methanocaldococcus jannaschii glutaminase
      Kumar, Sanjeev; Prakash, Sunita; Gupta, Kallol; Dongre, Aparna; Balaram, Padmanabhan; Balaram, Hemalatha
      Nature Communications (2016), 7 (), 12798CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      Protein ageing is often mediated by the formation of succinimide intermediates. These short-lived intermediates derive from asparaginyl deamidation and aspartyl dehydration and are rapidly converted into β-aspartyl or D-aspartyl residues. Here we report the presence of a highly stable succinimide intermediate in the glutaminase subunit of GMP synthetase from the hyperthermophile Methanocaldoccocus jannaschii. By comparing the biophys. properties of the wild-type protein and of several mutants, we show that the presence of succinimide increases the structural stability of the glutaminase subunit. The protein bearing this modification in fact remains folded at 100 °C and in 8 M guanidinium chloride. Mutation of the residue following the reactive asparagine provides insight into the factors that contribute to the hydrolytic stability of the succinimide. Our findings suggest that sequences that stabilize succinimides from hydrolysis may be evolutionarily selected to confer extreme thermal stability.
    56. 56
      Roth, C., Weizenmann, N., Bexten, N., Saenger, W., Zimmermann, W., Maier, T., and Strater, N. (2017) Amylose recognition and ring-size determination of amylomaltase. Sci. Adv. 3, 3,  DOI: 10.1126/sciadv.1601386
      There is no corresponding record for this reference.
    57. 57
      Acedo, J. Z., Bothwell, I. R., An, L., Trouth, A., Frazier, C., and van der Donk, W. A. (2019) O-Methyltransferase-Mediated Incorporation of a beta-Amino Acid in Lanthipeptides. J. Am. Chem. Soc. 141, 16790– 16801,  DOI: 10.1021/jacs.9b07396
      57
      O-Methyltransferase-Mediated Incorporation of a β-Amino Acid in Lanthipeptides
      Acedo, Jeella Z.; Bothwell, Ian R.; An, Linna; Trouth, Abby; Frazier, Clara; van der Donk, Wilfred A.
      Journal of the American Chemical Society (2019), 141 (42), 16790-16801CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Lanthipeptides represent a large class of cyclic natural products defined by the presence of lanthionine (Lan) and methyllanthionine (MeLan) cross-links. With the advances in DNA sequencing technologies and genome mining tools, new biosynthetic enzymes capable of installing unusual structural features are continuously being discovered. In this study, we investigated an O-methyltransferase that is a member of the most prominent auxiliary enzyme family assocd. with class I lanthipeptide biosynthetic gene clusters. Despite the prevalence of these enzymes, their function has not been established. Herein, we demonstrate that the O-methyltransferase OlvSA encoded in the olv gene cluster from Streptomyces olivaceus NRRL B-3009 catalyzes the rearrangement of a highly conserved aspartate residue to a β-amino acid, isoaspartate, in the lanthipeptide OlvA(BCSA). We elucidated the NMR soln. structure of the GluC-digested peptide, OlvA(BCSA)GluC, which revealed a unique ring topol. comprising four interlocking rings and positions the isoaspartate residue in a solvent exposed loop that is stabilized by a MeLan ring. Gas chromatog.-mass spectrometry anal. further indicated that OlvA(BCSA) contains two DL-MeLan rings and two Lan rings with an unusual LL-stereochem. Lastly, in vitro reconstitution of OlvSA activity showed that it is a leader peptide-independent and S-adenosyl methionine-dependent O-methyltransferase that mediates the conversion of a highly conserved aspartate residue in a cyclic substrate into a succinimide, which is hydrolyzed to generate an Asp or isoAsp contg. peptide. This overall transformation converts an α-amino acid into a β-amino acid in a ribosomally synthesized peptide, via an electrophilic intermediate that may be the intended product.
    58. 58
      Janetzko, J. and Walker, S. (2017) Aspartate Glycosylation Triggers Isomerization to Isoaspartate. J. Am. Chem. Soc. 139, 3332– 3335,  DOI: 10.1021/jacs.6b12866
      58
      Aspartate glycosylation triggers isomerization to isoaspartate
      Janetzko, John; Walker, Suzanne
      Journal of the American Chemical Society (2017), 139 (9), 3332-3335CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      O-linked β-N-acetylglucosamine transferase (OGT) is an essential human enzyme that glycosylates numerous nuclear and cytoplasmic proteins on Ser and Thr residues. It also cleaves host cell factor 1 (HCF-1) by a mechanism in which the 1st step involves glycosylation on Glu residues. Replacing Glu with Asp residues in an HCF-1 proteolytic repeat was shown to prevent peptide backbone cleavage, but whether Asp glycosylation occurred was not examd. Here, we report that OGT glycosylates Asp residues much faster than it glycosylates Glu residues in an otherwise identical model peptide substrate; moreover, once formed, the glycosylated Asp residue reacts further to form a succinimide intermediate that hydrolyzes to produce the corresponding isoaspartyl peptide. Asp to isoAsp residue isomerization in proteins occurs in cells, but was previously thought to be exclusively nonenzymic. These findings suggest it may also be enzyme-catalyzed. In addn. to OGT, enzymes that may catalyze Asp to isoAsp isomerization include poly(ADP-ribose) polymerases, enzymes known to ribosylate Asp residues in the process of poly(ADP-ribosyl)ation.
    59. 59
      Deverman, B. E., Cook, B. L., Manson, S. R., Niederhoff, R. A., Langer, E. M., Rosova, I., Kulans, L. A., Fu, X. Y., Weinberg, J. S., Heinecke, J. W., Roth, K. A., and Weintraub, S. J. (2002) Bcl-X-L deamidation is a critical switch in the regulation of the response to DNA damage. Cell 111, 51– 62,  DOI: 10.1016/S0092-8674(02)00972-8
      59
      Bcl-xL deamidation is a critical switch in the regulation of the response to DNA damage
      Deverman, Benjamin E.; Cook, Brian L.; Manson, Scott R.; Niederhoff, Robert A.; Langer, Ellen M.; Rosova, Ivana; Kulans, Laura A.; Fu, Xiaoyun; Weinberg, Justin S.; Heinecke, Jay W.; Roth, Kevin A.; Weintraub, Steven J.
      Cell (Cambridge, MA, United States) (2002), 111 (1), 51-62CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      The therapeutic value of DNA-damaging antineoplastic agents is dependent upon their ability to induce tumor cell apoptosis while sparing most normal tissues. Here, we show that a component of the apoptotic response to these agents in several different types of tumor cells is the deamidation of two asparagines in the unstructured loop of Bcl-xL, and we demonstrate that deamidation of these asparagines imports susceptibility to apoptosis by disrupting the ability of Bcl-xL to block the proapoptotic activity of BH3 domain-only proteins. Conversely, Bcl-xL deamidation is actively suppressed in fibroblasts, and suppression of deamidation is an essential component of their resistance to DNA damage-induced apoptosis. Our results suggest that the regulation of Bcl-xL deamidation has a crit. role in the tumor-specific activity of DNA-damaging antineoplastic agents.
    60. 60
      Lee, J. C., Kang, S. U., Jeon, Y., Park, J. W., You, J. S., Ha, S. W., Bae, N., Lubec, G., Kwon, S. H., Lee, J. S., Cho, E. J., and Han, J. W. (2012) Protein L-isoaspartyl methyltransferase regulates p53 activity. Nat. Commun. 3, 3,  DOI: 10.1038/ncomms1933
      There is no corresponding record for this reference.
    61. 61
      Curnis, F., Longhi, R., Crippa, L., Cattaneo, A., Dondossola, E., Bachi, A., and Corti, A. (2006) Spontaneous formation of L-isoaspartate and gain of function in fibronectin. J. Biol. Chem. 281, 36466– 36476,  DOI: 10.1074/jbc.M604812200
      61
      Spontaneous Formation of L-Isoaspartate and Gain of Function in Fibronectin
      Curnis, Flavio; Longhi, Renato; Crippa, Luca; Cattaneo, Angela; Dondossola, Eleonora; Bachi, Angela; Corti, Angelo
      Journal of Biological Chemistry (2006), 281 (47), 36466-36476CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Isoaspartate formation in extracellular matrix proteins, by aspartate isomerization or asparagine deamidation, is generally viewed as a degrdn. reaction occurring in vivo during tissue aging. For instance, non-enzymic isoaspartate formation at RGD-integrin binding sites causes loss of cell adhesion sites, which in turn can be enzymically "repaired" to RGD by protein-L-isoAsp-O-methyltransferase. We show here that isoaspartate formation is also a mechanism for extracellular matrix activation. In particular, we show that deamidation of Asn263 at the Asn-Gly-Arg (NGR) site in fibronectin N-terminal region generates an αvβ3-integrin binding site contg. the L-isoDGR sequence, which is enzymically "deactivated" to DGR by protein-L-isoAsp-O-methyltransferase. Furthermore, rapid NGR-to-isoDGR sequence transition in fibronectin fragments generates αvβ3 antagonists (named "isonectins") that competitively bind RGD binding sites and inhibit endothelial cell adhesion, proliferation, and tumor growth. Time-dependent generation of isoDGR may represent a sort of mol. clock for activating latent integrin binding sites in proteins.
    62. 62
      Mallagaray, A., Creutznacher, R., Dulfer, J., Mayer, P. H. O., Grimm, L. L., Orduna, J. M., Trabjerg, E., Stehle, T., Rand, K. D., Blaum, B. S., Uetrecht, C., and Peters, T. (2019) A post-translational modification of human Norovirus capsid protein attenuates glycan binding. Nat. Commun. 10, 10,  DOI: 10.1038/s41467-019-09251-5
      There is no corresponding record for this reference.
    63. 63
      Lindner, H., Sarg, B., Grunicke, H., and Helliger, W. (1999) Age-dependent deamidation of H1(0) histones in chromatin of mammalian tissues. J. Cancer Res. Clin. Oncol. 125, 182– 186,  DOI: 10.1007/s004320050261
      63
      Age-dependent deamidation of H1° histones in chromatin of mammalian tissues
      Lindner, Herbert; Sarg, Bettina; Grunicke, Hans; Helliger, Wilfried
      Journal of Cancer Research and Clinical Oncology (1999), 125 (3/4), 182-186CODEN: JCROD7; ISSN:0171-5216. (Springer-Verlag)
      The compn. of the H1° histone sub-fractions was examd. in rat and mouse tissues. Using reverse-phase HPLC and hydrophilic-interaction liq. chromatog. the authors have found that the relative proportions of all 4 forms of H1° differ from tissue to tissue and from species to species. In principle, the authors obsd. an age-dependent increase in the amt. of both the N-terminally acetylated (H1°a Asn-3 and H1°a Asp-3) and the deamidated forms of H1° (H°a Asp-3 and H1°b Asp-3). Compared with the proportion of N-terminally acetylated H1° forms in liver, kidney, and brain of rats and mice 20 days of age, the authors found an increase in these H1° subfractions of ≤30% in the corresponding organs of 300-day-old animals. The proportion at deamidated H1° forms was 1.6-4-fold higher in the livers and 8-12-fold higher in the brains of 300-day-old mice and rats, resp., than in 20-day-old animals. The tissue-specific nature of the ratio of H1° subfractions suggests that the different forms of histone H1° have specific individual functions. The possible biol. significance of age-related accumulation of N-terminal acetylated and deamidated histone H1° forms is discussed in the light of these results.
    64. 64
      David, C. L., Keener, J., and Aswad, D. W. (1999) Isoaspartate in ribosomal protein S11 of Escherichia coli. J. Bacteriol. 181, 2872– 2877,  DOI: 10.1128/JB.181.9.2872-2877.1999
      64
      Isoaspartate in ribosomal protein S11 of Escherichia coli
      David, Cynthia L.; Keener, John; Aswad, Dana W.
      Journal of Bacteriology (1999), 181 (9), 2872-2877CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      Isoaspartyl sites, in which an aspartic acid residue is linked to its C-flanking neighbor via its β-carboxyl side chain, are generally assumed to be an abnormal modification arising as proteins age. The enzyme protein L-isoaspartate methyltransferase (PIMT), present in many bacteria, plants, and animals, catalyzes the conversion of isoaspartate to normal α-linked aspartyl bonds and is thought to serve an important repair function in cells. Having introduced a plasmid into Escherichia coli that allows high-level expression of rat PIMT, we explored the possibility that the rat enzyme reduces isoaspartate levels in E. coli proteins, a result predicted by the repair hypothesis. The present study demonstrates that this is indeed the case; E. coli cells expressing rat PIMT had significantly lower isoaspartate levels than control cells, esp. in stationary phase. Moreover, the distribution of isoaspartate-contg. proteins in E. coli differed dramatically between logarithmic-and stationary-phase cultures. In stationary-phase cells, a no. of proteins in the mol. mass range of 66 to 14 kDa contained isoaspartate, whereas in logarithmic-phase cells, nearly all of the detectable isoaspartate resided in a single 14-kDa protein which we identified as ribosomal protein S11. The near stoichiometric levels of isoaspartate in S11, estd. at 0.5 mol of isoaspartate per mol of S11, suggests that this unusual modification may be important for S11 function.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biochem.0c00502.

    • Additional data and figures including reanalysis of MurA structures, AdoHcy calibration curve, analysis of the reversibility of chemical denaturation, protein characterization (size-exclusion chromatograms, SDS-PAGE and mass spectra), peptide characterization (LC-MS and MS/MS spectra), deamidation analysis (HPLC traces), peptide mapping, CD spectra, ion mobility mass spectra, and Woods plots for HDX experiments (PDF)

    Accession Codes

    P33038.


    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.