Download Hi-Res ImageDownload to MS-PowerPointCite This:Biochemistry 2014, 53, 15, 2454-2463

Detergent Optimized Membrane Protein Reconstitution in Liposomes for Solid State NMR

Dylan T. Murray^†^‡
,
James Griffin^‡^§
, and
Timothy A. Cross^*^†^‡^§

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† Institute for Molecular Biophysics, Florida State University, 91 Chieftan Way, Tallahassee, Florida 32306, United States

‡ The National High Magnetic Field Laboratory, 1800 E. Paul Dirac Dr., Tallahassee, Florida 32310, United States

§ Department of Chemistry and Biochemistry, Florida State University, 95 Chieftan Way, Tallahassee, Florida 32306, United States

*1800 E. Paul Dirac Drive, Tallahassee, FL, 32310. Phone: 850-644-0917. E-mail: [email protected]

Cite this: Biochemistry 2014, 53, 15, 2454–2463

Publication Date (Web):March 25, 2014

https://doi.org/10.1021/bi500144h

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Abstract

For small helical membrane proteins, their structures are highly sensitive to their environment, and solid state NMR is a structural technique that can characterize these membrane proteins in native-like lipid bilayers and proteoliposomes. To date, a systematic method by which to evaluate the effect of the solubilizing detergent on proteoliposome preparations for solid state NMR of membrane proteins has not been presented in the literature. A set of experiments are presented aimed at determining the conditions most amenable to dialysis mediated reconstitution sample preparation. A membrane protein from M. tuberculosis is used to illustrate the method. The results show that a detergent that stabilizes the most protein is not always ideal and sometimes cannot be removed by dialysis. By focusing on the lipid and protein binding properties of the detergent, proteoliposome preparations can be readily produced, which provide double the signal-to-noise ratios for both the oriented sample and magic angle spinning solid state NMR. The method will allow more membrane protein drug targets to be structurally characterized in lipid bilayer environments.

Membrane protein isolation and purification requires solubilization of the proteins in detergent micelles. Structure determination by solid state NMR in a native-like lipid bilayer requires the removal of these detergents during reconstitution into liposomes. The membrane protein bilayer or proteoliposome samples can be very sensitive to the presence of residual detergent. To date, a systematic detergent screen has not been described for the reconstitution of small helical membrane protein samples into lipid environments for solid state NMR.

Solid state NMR is responsible for the majority of helical membrane protein structures characterized in lipid bilayers. (1-7) Both oriented sample (OS) and magic angle spinning (MAS) samples are sensitive to the detergent used in purification and reconstitution. For OS solid state NMR, mechanically aligned samples rely on planar lipid bilayers, and consequently, the induced curvature from residual detergent molecules can be significant. (8) Alternative bicelle samples are very sensitive to the ratio of short to long chain lipids in the sample, (9) and an unintended detergent from a purification protocol would alter the phase behavior of bicelles. For MAS, the influence of residual detergent may not be immediately detectable in the spectra. However, recent studies have shown that sensitivity and resolution are both dependent on lipid type (10) and require the complete removal of detergent. (11) In addition, we have found that residual detergent significantly reduces the stability and hence lifetime of the samples. As the technique is applied to proteins with more than one or two helices, the affinity of detergent for liposomes and for the protein itself must be considered in the preparation of samples. Multispan helical membrane proteins are likely to have more detergent binding capacity in the hydrophobic region of the membrane than single helix membrane proteins. (12) It is beneficial then to have a distinct set of experiments by which to optimize the detergent choice for solid state NMR sample preparation.

Many detergent based screens have been performed in the context of membrane protein structural biology. Here, we focus on the use of detergents for reconstitution of purified proteins into liposomes. It is worthwhile noting that different considerations are applicable when solubilizing protein from cells, and these have been discussed in detail. (12) Crystallization screens for X-ray (13-15) and electron (16, 17) diffraction have focused on stabilizing proteins in 3D or 2D detergent and lipid lattices. Solution NMR efforts focus on achieving isotropic correlation times in detergent micelle preparations. (18-26) In all cases, there should be an emphasis on achieving a fully functional state for the membrane protein, a parameter that can vary greatly depending on the solubilizing conditions used. (27-29) Unfortunately, for many membrane proteins, such as ion channels, functional assays require a bilayer preparation. Lipid bilayer and preoliposome samples for solid state NMR permits both structural studies and functional assays in a native-like environment. However, it is often unclear if a residual detergent is present and if present whether or not it interferes with the protein structure and function. (30, 31) One of the distinguishing characteristics between detergents and lipids is the high monomer concentration of detergents in the presence of micelles that can lead to detergent binding in non-native locations, such as between helices (32, 33) or at the aqueous surface, (34) which could explain the loss of function for proteins in detergent environments. (35) Therefore, based on the potential impact on sample preparation and protein structure and function, we have developed a protocol to eliminate the residual detergent from the samples used for solid state NMR spectroscopy.

There have been studies of detergent affinity for liposomes (36-39) and of the kinetics of detergent insertion into liposomes. (40) Pure liposomes can be prepared with no residual detergent, (41) but the introduction of detergent-bound protein into a solution of liposomes can modify the detergent affinity for the liposomes and the kinetics for the detergent removal from the liposomes as a result of the known affinity of detergents for protein molecules. (12) While both long and short acyl chain detergents have stabilized a variety of membrane protein structures, long acyl chain detergents provide a better hydrophobic environment. (18, 23) Unfortunately, detergents, of a given headgroup structure, with longer acyl chains have greater affinity for and increased partitioning into liposomes than those with shorter acyl chains. (42) Therefore, a general theme for reconstitution based detergent screens would be to decrease the acyl chain length as much as possible to minimize the residual detergent in the final proteoliposome sample.

Here, we devise a detergent screen that aims to improve the sample quality for solid state NMR. The samples can be used for either OS solid state NMR of mechanically aligned lipid bilayers or MAS solid state NMR of proteoliposomes. Traditional batch purification (43) with detergent exchange (20) is used to transfer the protein into a new detergent. This can be followed by size exclusion chromatography and circular dichroism (CD) spectroscopy to initially evaluate the protein structure in the new environment. Continued characterization can be achieved with HPLC using evaporative light scattering detection (ELSD) for assessing the residual detergent in the proteoliposomes. We apply the screen here to a small three helix membrane protein, Rv1861 from M. tuberculosis, that hydrolyzes nucleotides when reconstituted into proteoliposomes and participates in the regulation of transglycosylase activity. OS solid state NMR spectra of this protein have been published for samples prepared using two different detergents. (44, 45) The results of this work indicate that a detergent with moderate affinity for liposomes can sufficiently solubilize the protein and be completely removed during reconstitution allowing for increased sensitivity in the solid state NMR samples. On the basis of the results presented here, we expect that many more helical membrane proteins can be readily prepared in liposomes for solid state NMR studies.

Experimental Procedures

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Protein Expression and Batch Purification

The Rv1861 gene from M. tuberculosis strain H37Rv was cloned into a modified pET-16b vector (Novagen, Inc.) modified to include a His₆ N-terminal purification tag. The plasmid was transformed into E. coli BL21-RP-Codon Plus cells (Stratagene, Inc.) for expression. Cells were grown in LB media (Ameresco, Inc.) at 37 °C to an OD₆₀₀ of 1.0 before expression was induced by adding IPTG to 0.4 mM. Cells were harvested, and 10 mL of lysis buffer (75 mM sodium phosphate, pH 7.5, and 500 mM sodium chloride) per unit of OD₆₀₀ absorbance was added to resuspend the cells. Lysozyme was added to 0.25 mg/mL along with 4 μL benzonase nuclease and cells incubated at room temperature for 30 min prior to French Press at 10,000 PSI three times. Ten milliliters of lysate was centrifuged at 18,000g for 60 min to isolate the inclusion bodies from other cellular components. Six milliliters of solubilization buffer (40 mM sodium phosphate, pH 7.5, 300 mM sodium chloride, 108 mM Empigen-BB) was used to resuspend each pellet, which was then incubated at 4 °C with gentle rocking for 4 h. The suspension was centrifuged at 18,000g for 30 min to remove insoluble material.

Eight purification tests were prepared by adding 700 μL of the supernatant to 100 μL of Qiagen Ni-NTA resin equilibrated with equilibration buffer (40 mM sodium phosphate, pH 7.5, 300 mM sodium chloride, and 25 mM Empigen-BB) in a 1.5 mL Eppendorf tube. The mixtures were incubated at room temperature for 4 h to allow the proteins to bind resin. The tubes were centrifuged at 1,000g for 1 min, and the supernatant was removed without disturbing the resin. Then 500 μL of equilibration buffer was added to each Eppendorf tube and the mixture incubated at room temperature with gentle rocking for 5 min. Next, 500 μL of wash buffer (equilibration buffer with 20 mM imidazole) was added and the tubes incubated and centrifuged as before. The processes was repeated twice more, once with wash buffer, then once with exchange buffer (20 mM sodium phosphate at pH 7.5 with a quantity of detergent expected to yield 0.4 mM concentration of micelles (Tables 1 and 2)). Protein was eluted from the resin by repeating the process three more times with elution buffer (exchange buffer with 500 mM imidazole.)

Table 1. Detergent Concentrations Used for the Batch Purification Assay

detergent	concn (mM)
sodium dodecylsulfate	33
Empigen-BB	25
dodecylphosphocholine	19
sodium dodecylsarcosine	41
decyldimethylglycine	43
Anzergent 3–10	55
Anzergent 3–8	414
nonylglucoside	12
nonylmaltoside	39

Table 2. Detergent Parameters in Aqueous Solutiona

detergent	molecular weight (Da)	CMC (mM)	aggregation number	micelle mass (kDa)	acyl chain	headgroup charge
SDSb	289	7–10 (0.5)d	62	18	12	negative
Empigen-BBb	272	1.6–2.1	*	*	12e	zwitterionic
DPCc	351	1.5	54	19	12	zwitterionic
Sarcoc	293	14.4	*	*	12	negative
DMc	483	1.8	69	33	10	polar
DDGlyc	243	19	*	*	10	zwitterionic
A3–10c	308	39	41	13	10	zwitterionic
NMc	469	6	25	12	9	polar
NGc	306	6.5	133	41	9	polar
OGc	292	18–20	27–100	8–29	8	polar
A3–8c	280	390	*	*	8	zwitterionic

An asterisk indicates that data was not available.

Values obtained from Sigma-Aldrich, Inc.

Values obtained from Anatrace/Affymetrix, Inc.

SDS–CMC in the presence of proteins is reduced to ∼0.5 mM. (53)

Empigen-BB is primarily 12 carbon but contains 10–16 carbon molecules.

Size Exclusion Chromatography

A Hi-Prep Sephacryl S200 (16/60) 120 mL column (GE Lifesciences, Inc.) and AKTA Xpress (GE Lifesciences, Inc.) system were used to perform size exclusion chromatography at room temperature. For each detergent, ∼20 μL of eluted protein was diluted to 500 μL with exchange buffer (see Protein Expression and Batch Purification section for buffer contents) and concentrated to ∼100 μL using a 3.5 kDa spin column (Millipore, Inc.). The dilution and concentration procedure was repeated once more before harvesting the solubilized protein and diluting it to 5 mL to remove imidazole from the solution that would interfere with the UV detection of the protein and to prepare the sample for injection. The column was equilibrated with two column volumes of size exclusion buffer (20 mM sodium phosphate at pH 7.5, with detergent at the critical micelle concentration (CMC); see Table 2). The protein sample was then injected onto the column, followed by a 5 mL wash with exchange buffer to ensure that the entire sample was removed from the injection loop. The column was run at 0.5 mL/min for 140 mL.

CD Spectroscopy

Purified protein was rinsed three times in 500 μL, 3.5 kDa spin filters (Millipore, Inc.) with 25 mM sodium phosphate at pH 7.5 and a concentration of 1 CMC of the appropriate detergent (Table 2) to remove imidazole. The protein was diluted to between 5 and 10 μM for CD experiments depending on the detergent used. The samples were desalted with data acquired in a 300 μL, 1 mm, Starna quartz cell on an AVIV 202 CD spectrometer. Three sweeps of the 180–260 nm wavelength range in 1.0 nm increments were averaged, and the buffer signal was subtracted from the protein signal before converting the raw data into residual molar ellipticity. The deconvolution of the CD curves was performed in CD-PRO (http://lamar.colostate.edu/∼sreeram/CDPro/main.html) using a basis set of 43 soluble and 13 membrane proteins. The CD data were deposited in the PDCDB with codes CD0004475000, CD0004476000, CD0004477000, CD0004478000, and CD0004479000.

Proteoliposome Preparation

For the reconstitution test and the OS solid state NMR samples, a liposome suspension was prepared by dissolving 50 mg of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPC/DMPG) powder at a 4:1 weight ratio in 2 mL of 10 mM HEPES buffer at pH 7.5, in a glass test tube. The mixture was bath sonicated for 30–60 min until the solution became clear indicating the formation of small unilamellar vesicles, which was confirmed by electron microscopy (Figure 1, Supporting Information). The detergent was added to the solution until it was optically clear (0 absorbance at 0.1 cm path length over 300–700 nm), and all lipid vesicles were solubilized into mixed micelles (69 mM for sodium dodecyl sulfate (SDS) and 103 mM for decyl-N,N-dimethylglycine (DDGly)). Then, 4.2 mg (SDS) or 10.5 mg (DDGly) of detergent solubilized protein was added to the lipid detergent solution and the volume adjusted with 10 mM HEPES at pH 7.5 to a final volume of 8 mL. The detergent was added to make concentrations of 69 mM and 82 mM for SDS and DDGly, respectively, and the final lipid concentration was 9 mM. The mixture was incubated at 37 °C for 12 h. During incubation, the solution was mixed by gentle inversion three times at 4 h intervals. The solutions were placed in 6–8 kDa MWCO dialysis tubing and dialyzed against 2 L of 10 mM HEPES at pH 7.5 and 35 °C with daily buffer changes. Proteoliposomes were harvested after 10 d (SDS) or 6 d (DDGly) of dialysis by centrifugation at 228,000g for 1.5 h. The supernatant was discarded and the pellet resuspended with 10 mM HEPES at pH 7.5 to a final volume of 1.2 mL.

For magic angle spinning samples, the same reconstitution procedure was used except the lipid mass was reduced to 20 mg. The protein-to-lipid ratio remained the same for each detergent (1.8 mg for SDS and 4.2 mg for DDGly). After dialysis, the proteoliposomes were was pelleted at 228,000g for 2 h in 3.2 mL thickwall polycarbonate ultracentrifuge tubes (Beckman Coulter, Inc.)

Evaporative Light Scattering Detection

The detergents were separated using HPLC and an Acclaim Surfactant Plus 3.0 μm column (Thermo Scientific, Inc.). An AB linear gradient elution was used (30 °C, 70 to 15% eluent B over 10 min at a flow rate of 0.9 mL/min). Eluent A was HPLC grade acetonitrile, and eluent B was 0.1 M ammonium acetate at pH 5.0. The presence of the detergent was assessed using an ELSD380 system (Agilent Technologies, Inc.). After each daily dialysis buffer change, an aliquot of the detergent–proteoliposome mixture was removed from the dialysis bag. The aliquots were not diluted prior to each 25 μL injection. The N₂ flow rate was 1.6 L/min, the nebulizer was 50 °C, the evaporator was 75 °C, and the photomultiplier tube gain was set at 2.5 to ensure a noise level of <0.2 mV peak–peak based on manufacturer recommendations.

Oriented Sample Solid State NMR Samples and Spectroscopy

Proteoliposome solution was deposited on 40 5.7 mm × 12 mm × 60 μm glass slides (35 μL per slide). The slides were dehydrated in a sealed 98% relative humidity chamber at 22 °C for approximately 12 h or until bulk water was visibly removed from the slides. Two microliters of deionized water was added to the center of each slide before stacking 30 of them on top of each other. The stack was incubated at 98% relative humidity at 37 °C for 4 d to remove bulk water and to let the proteoliposome solution between the glass slides become homogenously hydrated. The stack was then inserted into a 5.7 mm × 5.7 mm × 20 mm glass cell and sealed with beeswax after inserting a plastic plug. (46) SAMPI4 (47) experiments for measuring ¹⁵N chemical shift anisotropy and ¹H–¹⁵N dipolar couplings were performed on a 21.1 T 105 mm bore magnet using a home-built Low-E ¹H-X static probe (48) at 310 K. A 4 μs ¹H pulse was used, and rf fields were 62.5 kHz on both channels for cross-polarization and ¹H decoupling. Thirty-two t₁ points were acquired with 2048 or 4096 transients averaged for DDGly and SDS, respectively. A 5 s recycle delay was used for both experiments.

Magic Angle Spinning Solid State NMR Samples and Spectroscopy

Proteoliposome pellets were dehydrated at 37 °C and 16% relative humidity for several hours until the sample was approximately 40% w/w water before packing into a 3.2 mm thin walled rotor (Revolution NMR, Inc.). The detailed packing procedure is described in ref 46. Dipolar assisted rotational resonance (DARR) (49, 50) experiments were performed on a 14.1 T 89 mm bore magnet using a home-built, Low-E, ¹H-¹³C-¹⁵N triple resonance probe (51) with 10 kHz MAS at 243 K. One hundred kilo hertz of irradiation on the ¹H channel was used for the 90° pulse and SPINAL-64 decoupling. (52) During cross-polarization, the ¹³C rf field was 50 kHz with a ±10% linear ¹H ramp. The ¹³C 90° pulse was 50 kHz. Sixty-four and 128 transients were averaged for each of the 512 t₁ points for DDGly and SDS, respectively. Recycle delay was set at 1.5 s.

Results

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Detergent Choice

It is important to distinguish between detergents used for isolation and purification and those used for reconstitution. When a preparation of purified protein solubilized in detergent is of interest, the detergent most amenable to the experiment being performed is often chosen. For example, the Rv1861 protein is easily purified in detergents with 12 carbon acyl chains (SDS and DPC) with SDS being the most suitable for solution NMR studies of this protein. (20) However, when the protein is reconstituted into liposomes attention must be paid to the detergent affinity for liposomes and protein. Ideally, this affinity should be minimized. The detergent can be exchanged on-column during the purification process once impurities have been removed. (20, 46) Rv1861 has been studied by OS solid state NMR in DMPC/DMPG lipid bilayers. The first samples were prepared using SDS mediated reconstitution. (44) The highest protein-to-lipid molar ratio (P/L) that could be used to prepare high quality mechanically aligned bilayer samples was 1:200. With less lipid per protein, the reconstitutions were incomplete, and the resuspended proteoliposome pellet was too viscous for NMR sample preparation. On the basis of this, we proposed that residual SDS was present in the samples, preventing high quality proteoliposome preparation. The most likely detergent characteristics to induce increased affinity for the proteoliposomes are acyl chain length and headgroup charge. More mild detergents lacking charge and containing shorter acyl chains, such as DM and OG, failed to stabilize the protein long enough for reconstitution into proteoliposomes. The detergent acyl chain length modulates the CMC for a given detergent headgroup (42) and is important during dialysis because only detergent monomers exchange with buffer. A higher CMC provides a greater concentration gradient and drives the detergent out of the proteoliposome and into the buffer. Furthermore, acyl chain length also determines relative affinity of the detergents for liposomes (42) and dictates how much detergent dissociates from the liposomes during dialysis. On the basis of the structure of SDS, we selected detergents with reduced acyl chain length (9–10 carbons) or a reduction in headgroup hydrophilicity under the premise that such detergents might stabilize the protein adequately and also be readily removed during reconstitution. The commercially available detergents selected for Rv1861 are shown in Figure 1 and Table 2.

Figure 1. Detergents used in the purification screen for Rv1861. Decreasing the acyl chain length for a given headgroup increases the relative CMC and decreases relative detergent affinity for liposomes. The dashed line is meant to suggest the approximate interface between hydrophilic and hydrophobic moieties for these amphipathic molecules. Molecular structures were drawn with ChemSketch (ACD/Laboratories, Inc.).

Batch Purification Assay

A batch purification assay was performed using the new detergents (Figure 2). Inclusion body fractions were solubilized with the industrial detergent Empigen-BB, bound to a Ni²⁺ affinity column, and washed in Empigen-BB followed by detergent exchange with the desired detergent and elution at a high concentration of imidazole. While SDS eluted the most protein, the other 8, 9, 10, and 12 carbon detergents eluted considerable protein. The 10 and 12 carbon detergents stabilized the protein for over 2 weeks, while the 8 and 9 carbon detergents all showed visible signs of precipitation within the same period. It is important to note that the >15 kDa bands visible in the InVision stained gel (Figure 2B) are most likely not the Rv1861 protein. The smaller molecular weight band around 16 kDa is not a dimer of Rv1861 whose molecular weight is 11.4 kDa. Also, both bands elute in either the flow through or wash fractions, indicating that these molecules do not have significant affinity for the Ni²⁺ column. Finally, the detergent A3–8 shows signs of protein precipitation or severe protein aggregation at the top of the gels as well as slightly increased molecular weight for the monomer (Figure 2A).

Oligomeric State in Micelles

Gel filtration chromatography was performed to assess sample homogeneity and to detect oligomeric states for the protein in the various detergent environments (Figure 3). The data are interpreted based on the standards provided by the manufacturer and the average micellar weight for each detergent. When no data were available for a detergent aggregation number, the value was estimated using data for similar detergents. Here, the detergents tested have micelle sizes spanning ∼7–20 kDa. It should be noted that the detergent contribution to molecular weight per protein molecule for oligomeric complexes can be expected to be somewhat less than that of a monomeric state; therefore, the influence from the detergent is not linear. The SDS sample eluted near the void volume of the column corresponding to a complex of ∼160 kDa. Note that the sample is heterogeneous with small peaks at 85 and 105 mL elution volumes. The DPC sample also eluted primarily as a large oligomeric complex of ∼100 kDa, although there is a small amount of a larger complex for this sample. The A3–10 sample elutes even later from the column and suggests a smaller complex of ∼60 kDa. There is a slight heterogeneity in the sample eluting at ∼55 mL. The DDGly solubilized protein eluted near the column volume (120 mL) and shows a homogeneous sample. This represents a monomeric complex (∼15–20 kDa) based on column standards and the molecular weight of Rv1861 (11.4 kDa). The SDS–PAGE results suggest monomeric states for Rv1861, while the SE results appear to show various oligomeric complexes for SDS, DPC, and A3–10. While the SDS–PAGE samples were not boiled, the higher concentration of SDS in the sample buffer could affect the protein conformation and oligomeric state.

Figure 3. Size exclusion elution profiles for selected detergents. SDS and DPC indicate the presence of a large oligomeric complex. A3–10 indicates the presence of an intermediate sized oligomeric complex, while DDGly most likely represents a monomeric complex. All detergents except DDGly have some heterogeneity in the sample. The detergent contribution to the molecular weight is most likely similar for all detergents, 7–20 kDa.

Alpha Helical Content in Micelles

CD spectroscopy was performed on the protein solubilized in detergents to assess helical content in the different environments. Helical prediction from the amino acid sequence using the TMHMM program (54) indicates the protein should be roughly 60% helical. The CD spectra all exhibit highly helical profiles (Figure 4) in agreement with TMHMM prediction and published OS solid state NMR data. (44, 45) The 12 carbon detergents exhibited similar secondary structure content. However, DDGly stabilized slightly more structures presenting slightly less unordered content, while A3–10 stabilized less α-helical content with a marked increase in the β-strand, turn, and unordered content.

Figure 4. (A) CD profiles for Rv1861 stabilized in various detergents. (B) Secondary structure content analysis of the CD data based on a database of 43 soluble and 13 membrane proteins.

Detergent Removal

Side-by-side reconstitution was performed into DMPC/DMPG lipid bilayers using protein stabilized in SDS or DDGly. Detergent was removed by dialysis with daily buffer changes. Figure 5A shows SDS–PAGE gels monitoring the reconstitution process. For the SDS mediated reconstitution, both detergent solubilized protein and proteoliposomes are present in the sample after extensive dialysis, although the amount of detergent solubilized protein is a small fraction of the total protein shown in Figure 5A. Lanes 1–3 are before dialysis and lanes 4–6 after dialysis with lane 4 being the supernatant after pelleting the proteoliposomes. Lanes 5 and 6 represent the proteoliposome pellet. There must be residual detergent in the sample to solubilize the hydrophobic membrane protein in the supernatant (lane 4). Conversely, the DDGly reconstitution does not show detergent solubilized protein in the supernatant after only three days of dialysis (Figure 5A, lane 4). This suggests that for DDGly virtually all of the detergent is removed by dialysis, while this is not true for SDS. For DDGly, the sample starts with a volume of 8 mL containing ∼80 mM DDGly and is equilibrated with 2 L of buffer for dialysis. After the first buffer change, there should be ∼0.3 mM DDGly remaining in the sample if complete equilibration occurs. By the third dilution, there should only be ∼5 nM of detergent left in the sample. Likewise, for SDS, the sample has an 8 mL volume with ∼70 mM detergent and is dialyzed in 2 L of buffer. Again, after three days the detergent concentration should be ∼5 nM if equilibrium is achieved. In the sample, the protein concentration is ∼0.1 mM, and the lipid concentration is ∼22 mM; therefore, if the detergent is equilibrated with each 2 L buffer change the remaining nanomolar concentration of detergent after 3 days should have no effect on the proteoliposomes because it is so dilute. The presence of detergent during dialysis was monitored by HPLC using an ELSD as shown in Figure 5B. Missing data points for SDS indicate the detector was saturated by large signals resulting from high concentrations of detergent. DDGly is undetectable after the third day of dialysis indicating the concentration is below the limit of detection (a few nanomolar) which verifies that the DDGly completely equilibrates with the buffer within 24 h. SDS, however, was still detected after 10 d indicating that the SDS did not equilibrate completely with buffer in 24 h since it was detectable even after the 10th buffer change. This result and the presence of solubilized protein in the supernatant from the ultracentrifugation suggest that a significant concentration of SDS remains in the sample.

Figure 5. (A) SDS–PAGE gels monitor the reconstitution process for Rv1861 in SDS and DDGly detergents show the residual soluble protein for the SDS sample. Lane 1 is purified protein in detergent micelles. Lanes 2 and 3 are detergent solubilized protein mixed with proteoliposomes before and after incubation at 37 °C, respectively. Lane 4–6 are samples after extensive dialysis and centrifugation. Lane 4 is the supernatant, and lanes 5 and 6 are the resuspended proteoliposome pellets before and after the removal of any precipitate. (B) Integrated peak volume for the detergent detected by evaporative light scattering as a function of dialysis time. The buffer with a 250-fold larger volume than the sample was changed daily. DDGly (squares) is readily removed but SDS (diamonds) persists in the sample after 10 daily buffer changes. SDS signals saturated the detector until day 6 due to high concentrations of the detergent. DDGly saturates the detector on day 0, but the sample was diluted. Measurement errors are ±5%.

Solid State NMR Sample Quality

OS solid state NMR samples prepared from SDS could only be made at 1:200 P/L, while DDGly can be prepared up to 1:80. The uniformly ¹⁵N-labeled SAMPI4 2D spectrum for each liposome preparation is shown in Figure 6. The highly congested spectrum is consistent with a three helix membrane protein. (45) A similar intensity pattern, such as the hole at 175 ppm chemical shift and 3.0 kHz dipolar coupling, indicates the protein has similar structure in each sample. However, in the boxed region of the spectrum there are resonances in the SDS spectrum not present in the DDGly sample (Figure 6). The exact structural changes cannot be determined with these spectra. Regardless, the higher P/L ratio available when using DDGly resulted in equivalent signal-to-noise with half of the signal averaging compared to that of the SDS sample.

Figure 6. SAMPI4 spectra of uniformly ¹⁵N-labeled Rv1861 in DMPC/DMPG lipid bilayers prepared using DDGly (A) and SDS (B). The spectra present similar intensity profiles indicating similar helical structure and orientation for both samples. The DDGly detergent allowed higher protein to lipid ratios to be used, which halved the signal averaging time for equivalent signal-to-noise. Contours are drawn at 1.1σ and 1.2σ with the factor between levels set to 1.1 for DDGly and SDS, respectively.

¹³C–¹³C magic angle spinning solid state NMR correlation spectra for Rv1861 proteoliposomes prepared using DDGly and SDS are shown in Figure 7. Because of the high content of hydrophobic amino acids, the resonances from these sites are not well resolved. However, both preparations give similar resonance envelopes for valine, leucine, and isoleucine, which are characteristic of helical membrane proteins. (55) Importantly, there are significant differences in the spectra. The SDS spectrum has missing threonine and tryptophan C_α/C_β cross-peaks (Figure 7, red arrows), and the DDGly spectrum has increased resolution (Figure 7, green arrows). The 1D slices shown in Figure 7C and D show narrower lineshapes, twice the signal-to-noise, and extra peaks for the DDGly sample.

Figure 7. ¹³C–¹³C DARR spectra (30 ms) for Rv1861 prepared from DDGly (A and C) and SDS (B and D) at 10 kHz MAS and 243 K. The SDS spectrum averaged twice as many transients as the DDGly spectrum but results in less signal. Red arrows indicate resonances missing from the SDS spectrum, and green arrows indicate cross-peaks for threonine, valine, and alanine that are better resolved for DDGly. Contours are drawn at 3.7σ and 7.8σ with the factor between levels set to 1.3 for DDGly and SDS, respectively.

Discussion

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Although a given detergent may stabilize a membrane protein, it does not mean that the detergent is the best candidate for the preparation of proteoliposome samples, as exemplified here with Rv1861. Detergents were screened based on the fact that short acyl chain detergents are easily removed from liposomes, while long acyl chain detergents are more difficult to remove from and have more affinity for protein molecules. For Rv1861, we show that reducing the acyl chain length from 12 to 10 allows for a doubling of the protein incorporated into liposomes while forming homogeneous samples without residual detergent. Furthermore, these preparations have been used for the first high resolution structural characterization of the transmembrane domain for a three helix membrane protein using OS and MAS solid state NMR (45) (Murray et al., unpublished results). On the basis of the set of experiments presented here, detergents can be screened for their effective removal leading to optimal samples of membrane protein drug targets for structural characterization by solid state NMR.

Every membrane protein interacts differently with each detergent. While some membrane proteins are solubilized very well in long, 12 carbon, acyl chain detergents, (56-58) others favor shorter, 8–10 carbon acyl chain detergents. (4, 12) In general, detergents with acyl chains containing <10 carbons are more easily removed unless there is a highly specific interaction with the protein. (12) However, in general, longer, 12 carbon acyl chain detergents are effective for stabilizing membrane proteins. (57, 59) A directed approach that is aimed at producing a native conformation in a native-like environment for solid state NMR is favored over stabilizing the greatest quantity of protein. As new membrane protein drug targets are studied by solid state NMR, the approach presented here represents an efficient strategy to begin structural characterization efforts. The lipid choice for the reconstitution of a specific protein is dependent on the successful results of a functional assay for each protein. For Rv1861, a GTP hydrolysis assay was used to show the protein is functional in DMPC/DMPG proteoliposomes.

It is completely plausible that the protein under study may be stabilized by a detergent in an inactive state with the wrong tertiary structure. Indeed, some proteins are completely inactivated by short exposure to detergents while remaining soluble, (35, 60, 61) and other proteins require cholesterol or specific lipids to achieve a similar activity in micelles as in liposomes, (58) and still others form nonfunctional oligomeric states. (62) The focus here is on identifying a detergent that will stabilize the protein for long enough to incorporate it into a native-like liposome environment while also allowing virtually all of the detergent to be removed by dialysis. The intended result is to obtain liposomes for solid state NMR with a high concentration of natively folded protein. It should be stressed that the screen applies to a variety of lipid combinations that can be tailored to the specific needs of a given protein and that the reconstituted proteins can be easily used in a variety of functional assays to validate the native-like character of the sample.

The experiments presented here provide a rapid method to screen detergents to optimize the incorporation of membrane proteins into liposomes. With the advent of solid state NMR technology as a primary technique for investigating membrane protein structure in a native-like environment, these experiments pave the way for many of these important drug targets to be characterized. Through this screening approach, many more membrane protein drug targets will become available for structural characterization.

Supporting Information

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Electron micrographs of the proteoliposome preparations obtained using SDS and DDGly mediated reconstitution. This material is available free of charge via the Internet at http://pubs.acs.org.

bi500144h_si_001.pdf (1.3 MB)

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Author Information

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Corresponding Author
- Timothy A. Cross - Institute for Molecular Biophysics, Florida State University, 91 Chieftan Way, Tallahassee, Florida 32306, United States; The National High Magnetic Field Laboratory, 1800 E. Paul Dirac Dr., Tallahassee, Florida 32310, United States; Department of Chemistry and Biochemistry, Florida State University, 95 Chieftan Way, Tallahassee, Florida 32306, United States; Email: [email protected]
Authors
- Dylan T. Murray - Institute for Molecular Biophysics, Florida State University, 91 Chieftan Way, Tallahassee, Florida 32306, United States; The National High Magnetic Field Laboratory, 1800 E. Paul Dirac Dr., Tallahassee, Florida 32310, United States
- James Griffin - The National High Magnetic Field Laboratory, 1800 E. Paul Dirac Dr., Tallahassee, Florida 32310, United States; Department of Chemistry and Biochemistry, Florida State University, 95 Chieftan Way, Tallahassee, Florida 32306, United States
Funding
The work was made possible by grants from the National Institute of Allergy and Infectious Diseases (R01-AI073891 and P01-AI074805) and the use of the National High Magnetic Field Laboratory, funded by a cooperative agreement between the State of Florida and the National Science Foundation (DMR-1157490).
Notes
The authors declare no competing financial interest.

Acknowledgment

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We thank Dr. Claudius Mondoma from the FSU Physical Biochemistry Facility, Institute for Molecular Biophysics for help with the CD experiments. Dr. Huajun Qin assisted with cloning and expression of the Rv1861 protein. Dr. Riqiang Fu, Dr. Bill Brey, and Peter Gor’kov from the NHMFL assisted with the NMR experiments and NMR probe technology.

Abbreviations

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A3–8	Anzergent 3–8
A3–10	Anzergent 3–10
CD	circular dichroism
CMC	critical micelle concentration
DARR	dipolar assisted rotational resonance
DMPC	1,2-dimyristoyl-sn-glycero-3-phosphocholine
DDGly	decyl-N,N-dimethylglycine
DMPG	1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol)
DPC	n-dodecylphosphocholine
Emp	Empigen-BB
ELSD	evaporative light scattering detection
HPLC	high pressure liquid chromatography
MAS	magic angle spinning
NM	n-nonly-β-d-glucopyranoside and n-nonly-β-d-maltropyranoside
OG	n-octyl-β-d-glucopyranoside
OS	oriented sample
Sarco	n-dodecanoyl sarcosine
SDS	sodium dodecyl sulfate

References

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The prepn. of high quality samples is a crit. challenge for the structural characterization of helical integral membrane proteins. Solving the structures of this diverse class of proteins by soln. NMR spectroscopy (NMR) requires that well-resolved 2D 1H/15N chem. shift correlation spectra be obtained. Acquiring these spectra demands the prodn. of samples with high levels of purity and excellent homogeneity throughout the sample. In addn., high yields of isotopically enriched protein and efficient purifn. protocols are required. We describe two robust sample prepn. methods for prepg. high quality, homogeneous samples of helical integral membrane proteins. These sample prepn. protocols have been combined with screens for detergents and sample conditions leading to the efficient prodn. of samples suitable for soln. NMR spectroscopy. We have examd. 18 helical integral membrane proteins, ranging in size from approx. 9 kDa to 29 kDa with 1-4 transmembrane helixes, originating from a no. of bacterial and viral genomes. 2D 1H/15N chem. shift correlation spectra acquired for each protein demonstrate well-resolved resonances, and >90% detection of the predicted resonances. These results indicate that with proper sample prepn., high quality soln. NMR spectra of helical integral membrane proteins can be obtained greatly enhancing the probability for structural characterization of these important proteins.
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Human peripheral-type cannabinoid receptor (CB2) was expressed in Escherichia coli as a fusion with the maltose-binding protein, thioredoxin, and a deca-histidine tag. Functional activity and structural integrity of the receptor in bacterial protoplast membranes was confirmed by extensive binding studies with a variety of natural and synthetic cannabinoid ligands. E. coli membranes expressing CB2 also activated cognate G-proteins in an in vitro coupled assay. Detergent-solubilized receptor was purified to 80%-90% homogeneity by affinity chromatog. followed by ion-exchange chromatog. By high-resoln. NMR on the receptor in DPC micelles, it was detd. that purified CB2 forms 1:1 complexes with the ligands CP-55,940 and anadamide. The receptor was successfully reconstituted into phosphatidylcholine bilayers and the membranes were deposited into a porous substrate as tubular lipid bilayers for structural studies by NMR and scattering techniques.
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Oliver, R. C., Lipfert, J., Fox, D. A., Lo, R. H., Doniach, S., and Columbus, L. (2013) Dependence of micelle size and shape on detergent alkyl chain length and head group PLoS One 8, e62488
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23
Dependence of micelle size and shape on detergent alkyl chain length and head group
Oliver, Ryan C.; Lipfert, Jan; Fox, Daniel A.; Lo, Ryan H.; Doniach, Sebastian; Columbus, Linda
PLoS One (2013), 8 (5), e62488CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a sol. protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are detd. for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2-1.5 Å per carbon addn. to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approx. const. for each detergent series. In addn., the aggregation no. of these detergents increases by ∼16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.
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Van Horn, W. D., Kim, H. J., Ellis, C. D., Hadziselimovic, A., Sulistijo, E. S., Karra, M. D., Tian, C., Sonnichsen, F. D., and Sanders, C. R. (2009) Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase Science 324, 1726– 1729
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25
Verardi, R., Shi, L., Traaseth, N. J., Walsh, N., and Veglia, G. (2011) Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method Proc. Natl. Acad. Sci. U.S.A. 108, 9101– 9106
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26
Yao, Y., Bobkov, A. A., Plesniak, L. A., and Marassi, F. M. (2009) Mapping the interaction of pro-apoptotic tBID with pro-survival BCL-XL Biochemistry 48, 8704– 8711
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27
Chiu, M. L., Tsang, C., Grihalde, N., and MacWilliams, M. P. (2008) Over-expression, solubilization, and purification of G protein-coupled receptors for structural biology Comb. Chem. High Throughput Screening 11, 439– 462
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27
Over-expression, solubilization, and purification of G protein-coupled receptors for structural biology
Chiu, Mark L.; Tsang, Cindy; Grihalde, Nelson; MacWilliams, Maria P.
Combinatorial Chemistry & High Throughput Screening (2008), 11 (6), 439-462CODEN: CCHSFU; ISSN:1386-2073. (Bentham Science Publishers Ltd.)
A review. With the advent of the recent detn. of high-resoln. crystal structures of bovine rhodopsin and human β2 adrenergic receptor (β2AR), there are still many structure-function relationships to be learned from other G protein-coupled receptors (GPCRs). Many of the pharmaceutically interesting GPCRs cannot be modeled because of their amino acid sequence divergence from bovine rhodopsin and β2AR. Structure detn. of GPCRs can provide new avenues for engineering drugs with greater potency and higher specificity. Several obstacles need to be overcome before membrane protein structural biol. becomes routine: over-expression, solubilization, and purifn. of milligram quantities of active and stable GPCRs. Coordinated iterative efforts are required to generate any significant GPCR over-expression. To formulate guidelines for GPCR purifn. efforts, we review published conditions for solubilization and purifn. using detergents and additives. A discussion of sample prepn. of GPCRs in detergent phase, bicelles, nanodiscs, or low-d. lipoproteins is presented in the context of potential structural biol. applications. In addn., a review of the solubilization and purifn. of successfully crystd. bovine rhodopsin and β2AR highlights tools that can be used for other GPCRs.
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Li, Q. X., Mittal, R., Huang, L. J., Travis, B., and Sanders, C. R. (2009) Bolaamphiphile-class surfactants can stabilize and support the function of solubilized integral membrane proteins Biochemistry 48, 11606– 11608
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29
Hanford, M. and Peeples, T. L. (2002) Archaeal tetraether lipids - unique structures and applications Appl. Biochem. Biotechnol. 97, 45– 62
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Archaeal tetraether lipids: Unique structures and applications
Hanford, Michael J.; Peeples, Tonya L.
Applied Biochemistry and Biotechnology (2002), 97 (1), 45-62CODEN: ABIBDL; ISSN:0273-2289. (Humana Press Inc.)
A review. The extremely stable biomols. manufd. by organisms from extreme environments are of great scientific and engineering interest in the development of robust and stable industrial biocatalysts. Identification of mols. that impart stability under extremes will also have a profound impact on our understanding of cellular survival. This review discusses isolation and characterization of archael tetraethers as well as target technologies for tetraether lipid application. The isolation and characterization of archaeal tetraether lipids has led to some interesting applications improving on ester lipid technologies. Potential applications include novel lubricants, gene-delivery systems, monolayer lipid matrixes for sensor devices, and protein stabilization. Following this review, patent abstrs. and addnl. literature pertaining to the isolation, characterization, and application of archaeal membrane lipids are listed.
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Ostuni, M. A., Iatmanen, S., Teboul, D., Robert, J. C., and Lacapere, J. J. (2010) Characterization of membrane protein preparations: measurement of detergent content and ligand binding after proteoliposomes reconstitution Methods Mol. Biol. 654, 3– 18
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Characterization of membrane protein preparations: measurement of detergent content and ligand binding after proteoliposomes reconstitution
Ostuni, Mariano A.; Iatmanen, Soria; Teboul, David; Robert, Jean-Claude; Lacapere, Jean-Jacques
Methods in Molecular Biology (New York, NY, United States) (2010), 654 (Membrane Protein Structure Determination), 3-18CODEN: MMBIED; ISSN:1064-3745. (Springer)
The study of membrane proteins is a difficult task due to their natural embedding in hydrophobic environment made by lipids. Solubilization and purifn. from native membranes or overexpressed system involves the use of detergent to make them sol. while maintaining their structural and functional properties. The choice of detergent is governed not only by their ability to reach these goals, but also by their compatibility with biochem. and structural studies. A different detergent can be used during purifn., and characterization of the detergent amts. present in each purifn. step is crucial. To address this point, we developed a colorimetric method to measure detergent content in different prepns. We analyzed detergent present in the collected fractions from the purifn. of the recombinant membrane translocator protein (RecTSPO). We followed detergent removal during the reconstitution of RecTSPO in liposomes and obsd. by electron microscopy the formation of proteoliposomes. We addressed the RecTSPO functionality by testing its ability to bind high affinity drug ligand [3H]PK 11195. We described the different parameters that should be controlled in order to optimize the measurement of this ligand binding using a filtration procedure. These protocols are useful to characterize functionality and detergent content of membrane protein, both key factors for further structural studies.
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Wang, L., Quan, C., Liu, B., Wang, J., Xiong, W., Zhao, P., and Fan, S. (2013) Functional reconstitution of staphylococcus aureus truncated agrc histidine kinase in a model membrane system PLoS One 8, e80400
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Zhou, H. X. and Cross, T. A. (2013) Influences of membrane mimetic environments on membrane protein structures Ann. Rev. Biophys. 42, 361– 392
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Influences of membrane mimetic environments on membrane protein structures
Zhou Huan-Xiang; Cross Timothy A
Annual review of biophysics (2013), 42 (), 361-92 ISSN:.
The number of membrane protein structures in the Protein Data Bank is becoming significant and growing. Here, the transmembrane domain structures of the helical membrane proteins are evaluated to assess the influences of the membrane mimetic environments. Toward this goal, many of the biophysical properties of membranes are discussed and contrasted with those of the membrane mimetics commonly used for structure determination. Although the mimetic environments can perturb the protein structures to an extent that potentially gives rise to misinterpretation of functional mechanisms, there are also many structures that have a native-like appearance. From this assessment, an initial set of guidelines is proposed for distinguishing native-like from nonnative-like membrane protein structures. With experimental techniques for validation and computational methods for refinement and quality assessment and enhancement, there are good prospects for achieving native-like structures for these very important proteins.
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Cross, T., Murray, D., and Watts, A. (2013) Helical membrane protein conformations and their environment Eur. Biophys. J. 42, 731– 755
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Helical membrane protein conformations and their environment
Cross, Timothy A.; Murray, Dylan T.; Watts, Anthony
European Biophysics Journal (2013), 42 (10), 731-755CODEN: EBJOE8; ISSN:0175-7571. (Springer)
A review. Evidence that membrane proteins respond conformationally and functionally to their environment is growing. Structural models, by necessity, have been characterized in prepns. where the protein has been removed from its native environment. Different structural methods have used various membrane mimetics that have recently included lipid bilayers as a more native-like environment. Structural tools applied to lipid bilayer-embedded integral proteins are informing us about important generic characteristics of how membrane proteins respond to the lipid environment as compared with their response to other nonlipid environments. Here, we review the current status of the field, with specific ref. to observations of some well-studied α-helical membrane proteins, as a starting point to aid the development of possible generic principles for model refinement.
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Zoonens, M., Comer, J., Masscheleyn, S., Pebay-Peyroula, E., Chipot, C., Miroux, B., and Dehez, F. (2013) Dangerous liaisons between detergents and membrane proteins. The case of mitochondrial uncoupling protein 2 J. Am. Chem. Soc. 135, 15174– 15182
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White, J. F. and Grisshammer, R. (2010) Stability of the neurotensin receptor NTS1 free in detergent solution and immobilized to affinity resin PLoS One 5, e12579
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36
Keller, M., Kerth, A., and Blume, A. (1997) Thermodynamics of interaction of octyl glucoside with phosphatidylcholine vesicles: partitioning and solubilization as studied by high sensitivity titration calorimetry Biochim. Biophys. Acta 1326, 178– 192
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de la Maza, A. and Parra, J. L. (1997) Solubilizing effects caused by the nonionic surfactant dodecylmaltoside in phosphatidylcholine liposomes Biophys. J. 72, 1668– 1675
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Tan, A., Ziegler, A., Steinbauer, B., and Seelig, J. (2002) Thermodynamics of sodium dodecyl sulfate partitioning into lipid membranes Biophys. J. 83, 1547– 1556
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Thermodynamics of sodium dodecyl sulfate partitioning into lipid membranes
Tan, Anmin; Ziegler, Andre; Steinbauer, Bernhard; Seelig, Joachim
Biophysical Journal (2002), 83 (3), 1547-1556CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
The partition equil. of sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate between water and bilayer membranes were investigated with isothermal titrn. calorimetry and spectroscopic methods (light scattering, 31P-NMR) in the temp. range of 28°C to 56°C. The partitioning of the dodecyl sulfate anion (DS-) into the bilayer membrane is energetically favored by an exothermic partition enthalpy of ΔHDo = -6.0 kcal/mol at 28°C. This is in contrast to nonionic detergents where ΔHDo is usually pos. The partition enthalpy decreases linearly with increasing temp. and the molar heat capacity is ΔCpo = -50 ± 3 cal mol-1 K-1. The partition isotherm is nonlinear if the bound detergent is plotted vs. the free detergent concn. in bulk soln. This is caused by the electrostatic repulsion between the DS- ions inserted into the membrane and those free in soln. near the membrane surface. The surface concn. of DS- immediately above the plane of binding was hence calcd. with the Gouy-Chapman theory, and a strictly linear relationship was obtained between the surface concn. and the extent of DS- partitioning. The surface partition const. K describes the chem. equil. in the absence of electrostatic effects. For the SDS-membrane equil. K was found to be 1.2 × 104 M-1 to 6 × 104 M-1 for the various systems and conditions investigated, very similar to data available for nonionic detergents of the same chain length. The membrane-micelle phase diagram was also studied. Complete membrane solubilization requires a ratio of 2.2 mol SDS bound per mol of total lipid at 56°C. The corresponding equil. concn. of SDS free in soln. is CD,fsol ∼ 1.7 mM and is slightly below the crit. micelles concn. (CMC) = 2.1 mM (at 56°C and 0.11 M buffer). Membrane satn. occurs at ∼ 0.3 mol SDS per mol lipid and the equil. SDS concn. is CD,fsat ≈ 2.2 mM ± 0.6 mM. SDS translocation across the bilayer is slow at ambient temp. but increases at high temps.
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Meister, A. and Blume, A. (2004) Solubilization of DMPC-d54 and DMPG-d54 vesicles with octylglucoside and sodium dodecyl sulfate studied by FT-IR spectroscopy Phys. Chem. Chem. Phys. 6, 1551– 1556
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Cócera, M., López, O., Estelrich, J., Parra, J. L., and de la Maza, A. (2000) Kinetic and structural aspects of the adsorption of sodium dodecyl sulfate on phosphatidylcholine liposomes Langmuir 16, 4068– 4071
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López, O., Cócera, M., Coderch, L., Parra, J. L., Barsukov, L., and de la Maza, A. (2001) Octyl glucoside-mediated solubilization and reconstitution of liposomes: structural and kinetic aspects J. Phys. Chem. B 105, 9879– 9886
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Maza, A., Lopez, O., Baucells, J., Gonzalez, P., and Parra, J. L. (1998) Solubilization of phosphatidylcholine unilamellar liposomes caused by alkyl glucosides J. Surfactants Deterg. 1, 381– 386
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Feng, G. and Winkler, M. E. (1995) Single-Step Purifications of His(6)-Muth, His(6)Mutl and His(6)-Muts Repair Proteins of Escherichia-Coli K-12 Biotechniques 19, 956– 965
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Li, C. G., Gao, P., Qin, H. J., Chase, R., Gor’kov, P. L., Brey, W. W., and Cross, T. A. (2007) Uniformly aligned full-length membrane proteins in liquid crystalline bilayers for structural characterization J. Am. Chem. Soc. 129, 5304– 5305
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Murray, D. T., Hung, I., and Cross, T. A. (2014) Assignment of oriented sample NMR resonances from a three transmembrane helix protein J. Magn. Reson. 240, 34– 44
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Das, N., Murray, D. T., and Cross, T. A. (2013) Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples Nat. Protoc. 8, 2256– 2270
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Nevzorov, A. A. and Opella, S. J. (2003) A “magic sandwich” pulse sequence with reduced offset dependence for high-resolution separated local field spectroscopy J. Magn. Reson. 164, 182– 186
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Gor’kov, P. L., Chekmenev, E. Y., Li, C. G., Cotten, M., Buffy, J. J., Traaseth, N. J., Veglia, G., and Brey, W. W. (2007) Using low-E resonators to reduce RF heating in biological samples for static solid-state NMR up to 900 MHz J. Magn. Reson. 185, 77– 93
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Takegoshi, K., Nakamura, S., and Terao, T. (2003) 13C–1H dipolar-driven 13C–13C recoupling without 13C rf irradiation in nuclear magnetic resonance of rotating solids J. Chem. Phys. 118, 2325– 2341
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Takegoshi, K., Nakamura, S., and Terao, T. (2001) 13C–1H dipolar-assisted rotational resonance in magic-angle spinning NMR Chem. Phys. Lett. 344, 631– 637
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McNeill, S. A., Gor’kov, P. L., Shetty, K., Brey, W. W., and Long, J. R. (2009) A low-E magic angle spinning probe for biological solid state NMR at 750 MHz J. Magn. Reson. 197, 135– 144
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Fung, B. M., Khitrin, A. K., and Ermolaev, K. (2000) An improved broadband decoupling sequence for liquid crystals and solids J. Magn. Reson. 142, 97– 101
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An Improved Broadband Decoupling Sequence for Liquid Crystals and Solids
Fung, B. M.; Khitrin, A. K.; Ermolaev, Konstantin
Journal of Magnetic Resonance (2000), 142 (1), 97-101CODEN: JMARF3; ISSN:1090-7807. (Academic Press)
Recently the authors developed an efficient broadband decoupling sequence called SPARC-16 for liq. crystals [J. Magn. Reson. 130, 317(1998)]. The sequence is based upon a 16-step phase cycling of the 2-step TPPM decoupling method for solids [J. Chem. Phys. 103, 6951(1995)]. Since then, a stepwise variation of the phase angle in the TPPM sequence offers even better results. The application of this new method to a liq. cryst. compd., 4-n-pentyl-4'-cyanobiphenyl, and a solid, l-tyrosine hydrochloride, is reported. The reason for the improvement is explained by an anal. of the problem in the rotating frame. (c) 2000 Academic Press.
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Ahmad, M. F., Ramakrishna, T., Raman, B., and Rao, C. M. (2006) Fibrillogenic and non-fibrillogenic ensembles of SDS-bound human α-synuclein J. Mol. Biol. 364, 1061– 1072
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Sonnhammer, E. L., von Heijne, G., and Krogh, A. (1998) A hidden Markov model for predicting transmembrane helices in protein sequences Proc.: Int. Conf. Intell. Syst. Mol. Biol. 6, 175– 182
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A hidden Markov model for predicting transmembrane helices in protein sequences
Sonnhammer E L; von Heijne G; Krogh A
Proceedings. International Conference on Intelligent Systems for Molecular Biology (1998), 6 (), 175-82 ISSN:1553-0833.
A novel method to model and predict the location and orientation of alpha helices in membrane-spanning proteins is presented. It is based on a hidden Markov model (HMM) with an architecture that corresponds closely to the biological system. The model is cyclic with 7 types of states for helix core, helix caps on either side, loop on the cytoplasmic side, two loops for the non-cytoplasmic side, and a globular domain state in the middle of each loop. The two loop paths on the non-cytoplasmic side are used to model short and long loops separately, which corresponds biologically to the two known different membrane insertions mechanisms. The close mapping between the biological and computational states allows us to infer which parts of the model architecture are important to capture the information that encodes the membrane topology, and to gain a better understanding of the mechanisms and constraints involved. Models were estimated both by maximum likelihood and a discriminative method, and a method for reassignment of the membrane helix boundaries were developed. In a cross validated test on single sequences, our transmembrane HMM, TMHMM, correctly predicts the entire topology for 77% of the sequences in a standard dataset of 83 proteins with known topology. The same accuracy was achieved on a larger dataset of 160 proteins. These results compare favourably with existing methods.
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Murray, D. T., Das, N., and Cross, T. A. (2013) Solid state NMR strategy for characterizing native membrane protein structures Acc. Chem. Res. 46, 2172– 2181
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Lund, S., Orlowski, S., Deforesta, B., Champeil, P., Lemaire, M., and Moller, J. V. (1989) Detergent structure and associated lipid as determinants in the stabilization of solubilized Ca-2+-ATPase from sarcoplasmic-reticulum J. Biol. Chem. 264, 4907– 4915
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Rosevear, P., VanAken, T., Baxter, J., and Ferguson-Miller, S. (1980) Alkyl glycoside detergents: a simpler synthesis and their effects on kinetic and physical properties of cytochrome c oxidase Biochemistry 19, 4108– 4115
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Alkyl glycoside detergents: a simpler synthesis and their effects on kinetic and physical properties of cytochrome c oxidase
Rosevear, Paul; VanAken, Terrell; Baxter, Jeffrey; Ferguson-Miller, Shelagh
Biochemistry (1980), 19 (17), 4108-15CODEN: BICHAW; ISSN:0006-2960.
Octyl glucoside (I) is an effective, nonionic, solubilizing agent for membrane proteins with the advantage of ease of removal by dialysis. In order to study the detergent-sensitive activity of cytochrome oxidase, I was chosen because of its simple structure and the possibility of synthesizing analogs to test the structural dependence of the detergent specificity. A procedure was therefore developed that facilitates large-scale prepn. of I and related alkyl glycosides, improving on previous methods by eliminating crystn. steps and employing a 1-step purifn. of the final product on Dowex 1. This new purifn. procedure is particularly important for achieving the level of purity required to obtain the disaccharide, longer alkyl chain detergents in sol. form. Of the alkyl glycosides prepd., lauryl (dodecyl) maltoside (II) was found to be the most successful as an activator of purified bovine and Neurospora cytochrome oxidases, giving 2-10-fold higher activities than I and other com. available detergents, Tween-20 and Triton X-100. Kinetic studies using 2 different steady-state assay systems indicate that the activity changes are not the result of altered binding of the substrate, but rather reflect a detergent effect on the state of assocn. of the enzyme (in a monomer, dimer, or polymer form). By gel filtration procedures, II and I were found to exist as monodisperse populations of micelles of 50,000 and 8000 daltons, resp. The small uniform micelles and chem. well-defined structures of II and I make them superior to other nonionic detergents for the study of membrane proteins in general, and cytochrome oxidase in particular, since its activity in lauryl II maltoside most closely approaches that of the physiol. state.
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Vukoti, K., Kimura, T., Macke, L., Gawrisch, K., and Yeliseev, A. (2012) Stabilization of functional recombinant cannabinoid receptor CB(2) in detergent micelles and lipid bilayers PLoS One 7, e46290
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Lowthert, L. A., Ku, N. O., Liao, J., Coulombe, P. A., and Omary, M. B. (1995) Empigen BB: a useful detergent for solubilization and biochemical analysis of keratins Biochem. Biophys. Res. Commun. 206, 370– 379
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Esmann, M. (1984) The Distribution of C12e8-Solubilized oligomers of the (Na+ + K+)-ATPase Biochim. Biophys. Acta 787, 81– 89
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Esmann, M., Skou, J. C., and Christiansen, C. (1979) Solubilization and molecular-weight determination of the (Na++K+)-ATPase from rectal glands of Squalus-acanthias Biochim. Biophys. Acta 567, 410– 420
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White, J. F., Grodnitzky, J., Louis, J. M., Trinh, L. B., Shiloach, J., Gutierrez, J., Northup, J. K., and Grisshammer, R. (2007) Dimerization of the class A G protein-coupled neurotensin receptor NTS1 alters G protein interaction Proc. Natl. Acad. Sci. U.S.A. 104, 12199– 12204
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Dimerization of the class A G protein-coupled neurotensin receptor NTS1 alters G protein interaction
White, Jim F.; Grodnitzky, Justin; Louis, John M.; Trinh, Loc B.; Shiloach, Joseph; Gutierrez, Joanne; Northup, John K.; Grisshammer, Reinhard
Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (29), 12199-12204, S12199/1-S12199/7CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
G protein-coupled receptors (GPCRs) have been found as monomers but also as dimers or higher-order oligomers in cells. The relevance of the monomeric or dimeric receptor state for G protein activation is currently under debate for class A rhodopsin-like GPCRs. Clarification of this issue requires the availability of well defined receptor prepns. as monomers or dimers and an assessment of their ligand-binding and G protein-coupling properties. We show by pharmacol. and hydrodynamic expts. that purified neurotensin receptor NTS1, a class A GPCR, dimerizes in detergent soln. in a concn.-dependent manner, with an apparent affinity in the low nanomolar range. At low receptor concns., NTS1 binds the agonist neurotensin with a Hill slope of ≈1; at higher receptor concns., neurotensin binding displays pos. cooperativity with a Hill slope of ≈2. NTS1 monomers activate Gαqβ1γ2, whereas receptor dimers catalyze nucleotide exchange with lower affinity. Our results demonstrate that NTS1 dimerization per se is not a prerequisite for G protein activation.
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Figure 1
Figure 1. Detergents used in the purification screen for Rv1861. Decreasing the acyl chain length for a given headgroup increases the relative CMC and decreases relative detergent affinity for liposomes. The dashed line is meant to suggest the approximate interface between hydrophilic and hydrophobic moieties for these amphipathic molecules. Molecular structures were drawn with ChemSketch (ACD/Laboratories, Inc.).
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Figure 2
Figure 2. Batch purification assay. SDS eluted the most protein, while other detergents eluted slightly less protein. The dashed line indicates detergents that failed to stabilize the protein for longer than two weeks. (A) Coomassie and (B) a UV sensitive poly histidine dye (InVision) were used to stain the gels. Inclusion Body is the resuspended pellet after spinning the lysate at 18,000g, Insoluble is the 18,000g pellet after incubation with the Empigen-BB detergent, Load is the supernatant from the insoluble pellet, Flow Through is the elute from the column with no imidazole present, Wash is the elute from the column with 40 mM imidazole, and all other lanes are the elutes from the column with 250 mM imidazole containing the detergent named at the top of each lane.
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Figure 3
Figure 3. Size exclusion elution profiles for selected detergents. SDS and DPC indicate the presence of a large oligomeric complex. A3–10 indicates the presence of an intermediate sized oligomeric complex, while DDGly most likely represents a monomeric complex. All detergents except DDGly have some heterogeneity in the sample. The detergent contribution to the molecular weight is most likely similar for all detergents, 7–20 kDa.
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Figure 4
Figure 4. (A) CD profiles for Rv1861 stabilized in various detergents. (B) Secondary structure content analysis of the CD data based on a database of 43 soluble and 13 membrane proteins.
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Figure 5
Figure 5. (A) SDS–PAGE gels monitor the reconstitution process for Rv1861 in SDS and DDGly detergents show the residual soluble protein for the SDS sample. Lane 1 is purified protein in detergent micelles. Lanes 2 and 3 are detergent solubilized protein mixed with proteoliposomes before and after incubation at 37 °C, respectively. Lane 4–6 are samples after extensive dialysis and centrifugation. Lane 4 is the supernatant, and lanes 5 and 6 are the resuspended proteoliposome pellets before and after the removal of any precipitate. (B) Integrated peak volume for the detergent detected by evaporative light scattering as a function of dialysis time. The buffer with a 250-fold larger volume than the sample was changed daily. DDGly (squares) is readily removed but SDS (diamonds) persists in the sample after 10 daily buffer changes. SDS signals saturated the detector until day 6 due to high concentrations of the detergent. DDGly saturates the detector on day 0, but the sample was diluted. Measurement errors are ±5%.
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Figure 6
Figure 6. SAMPI4 spectra of uniformly ¹⁵N-labeled Rv1861 in DMPC/DMPG lipid bilayers prepared using DDGly (A) and SDS (B). The spectra present similar intensity profiles indicating similar helical structure and orientation for both samples. The DDGly detergent allowed higher protein to lipid ratios to be used, which halved the signal averaging time for equivalent signal-to-noise. Contours are drawn at 1.1σ and 1.2σ with the factor between levels set to 1.1 for DDGly and SDS, respectively.
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Figure 7
Figure 7. ¹³C–¹³C DARR spectra (30 ms) for Rv1861 prepared from DDGly (A and C) and SDS (B and D) at 10 kHz MAS and 243 K. The SDS spectrum averaged twice as many transients as the DDGly spectrum but results in less signal. Red arrows indicate resonances missing from the SDS spectrum, and green arrows indicate cross-peaks for threonine, valine, and alanine that are better resolved for DDGly. Contours are drawn at 3.7σ and 7.8σ with the factor between levels set to 1.3 for DDGly and SDS, respectively.
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References
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  Stokes, David L.; Ubarretxena-Belandia, Iban; Gonen, Tamir; Engel, Andreas
  Methods in Molecular Biology (New York, NY, United States) (2013), 955 (Electron Crystallography of Soluble and Membrane Proteins), 273-296CODEN: MMBIED; ISSN:1064-3745. (Springer)
  A review. Membrane proteins play a tremendously important role in cell physiol. and serve as a target for an increasing no. of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex phys. chem. assocd. with membrane proteins has made them more difficult to study than their sol. cousins. Electron crystallog. has historically been a successful method for solving membrane protein structures and has the advantage of providing a native lipid environment for these proteins. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, electron microscopy can be used to collect images and diffraction and the corresponding data can be combined to produce a three-dimensional reconstruction, which under favorable conditions can extend to at. resoln. Like X-ray crystallog., the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallog., high-throughput methods for screening crystn. trials for electron crystallog. are not in general use. In this chapter, we describe two alternative methods for high-throughput screening of membrane protein crystn. within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the std. 96-well format allows the use of a liq.-handling robot and greatly increases throughput. The second method relies on titrn. of cyclodextrin as a chelating agent for detergent; a specialized pipetting robot has been designed not only to add cyclodextrin in a systematic way, but to use light scattering to monitor the reconstitution process. In addn., the use of liq.-handling robots for making neg. stained grids and methods for automatically imaging samples in the electron microscope are described.
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18. 18
  Columbus, L., Lipfert, J., Jambunathan, K., Fox, D. A., Sim, A. Y. L., Doniach, S., and Lesley, S. A. (2009) Mixing and matching detergents for membrane protein NMR structure determination J. Am. Chem. Soc. 131, 7320– 7326
  [ACS Full Text ], [CAS], Google Scholar
  18
  Mixing and Matching Detergents for Membrane Protein NMR Structure Determination
  Columbus, Linda; Lipfert, Jan; Jambunathan, Kalyani; Fox, Daniel A.; Sim, Adelene Y. L.; Doniach, Sebastian; Lesley, Scott A.
  Journal of the American Chemical Society (2009), 131 (21), 7320-7326CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
  One major obstacle to membrane protein structure detn. is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by NMR and ESR spectroscopy and small-angle x-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein's hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure detn. by NMR.
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19. 19
  Krueger-Koplin, R. D., Sorgen, P. L., Krueger-Koplin, S. T., Rivera-Torres, A. O., Cahill, S. M., Hicks, D. B., Grinius, L., Krulwich, T. A., and Girvin, M. E. (2004) An evaluation of detergents for NMR structural studies of membrane proteins J. Biomol. NMR 28, 43– 57
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20. 20
  Page, R. C., Moore, J. D., Nguyen, H. B., Sharma, M., Chase, R., Gao, F. P., Mobley, C. K., Sanders, C. R., Ma, L., Sonnichsen, F. D., Lee, S., Howell, S. C., Opella, S. J., and Cross, T. A. (2006) Comprehensive evaluation of solution nuclear magnetic resonance spectroscopy sample preparation for helical integral membrane proteins J. Struct. Funct. Genomics 7, 51– 64
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  20
  Comprehensive evaluation of solution nuclear magnetic resonance spectroscopy sample preparation for helical integral membrane proteins
  Page, Richard C.; Moore, Jacob D.; Nguyen, Hau B.; Sharma, Mukesh; Chase, Rose; Gao, Fei Philip; Mobley, Charles K.; Sanders, Charles R.; Ma, Liping; Soennichsen, Frank D.; Lee, Sangwon; Howell, Stanley C.; Opella, Stanley J.; Cross, Timothy A.
  Journal of Structural and Functional Genomics (2006), 7 (1), 51-64CODEN: JSFGAW; ISSN:1345-711X. (Springer)
  The prepn. of high quality samples is a crit. challenge for the structural characterization of helical integral membrane proteins. Solving the structures of this diverse class of proteins by soln. NMR spectroscopy (NMR) requires that well-resolved 2D 1H/15N chem. shift correlation spectra be obtained. Acquiring these spectra demands the prodn. of samples with high levels of purity and excellent homogeneity throughout the sample. In addn., high yields of isotopically enriched protein and efficient purifn. protocols are required. We describe two robust sample prepn. methods for prepg. high quality, homogeneous samples of helical integral membrane proteins. These sample prepn. protocols have been combined with screens for detergents and sample conditions leading to the efficient prodn. of samples suitable for soln. NMR spectroscopy. We have examd. 18 helical integral membrane proteins, ranging in size from approx. 9 kDa to 29 kDa with 1-4 transmembrane helixes, originating from a no. of bacterial and viral genomes. 2D 1H/15N chem. shift correlation spectra acquired for each protein demonstrate well-resolved resonances, and >90% detection of the predicted resonances. These results indicate that with proper sample prepn., high quality soln. NMR spectra of helical integral membrane proteins can be obtained greatly enhancing the probability for structural characterization of these important proteins.
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21. 21
  Shenkarev, Z. O., Lyukmanova, E. N., Paramonov, A. S., Shingarova, L. N., Chupin, V. V., Kirpichnikov, M. P., Blommers, M. J. J., and Arseniev, A. S. (2010) Lipid-protein nanodiscs as reference medium in detergent screening for high-resolution NMR studies of integral membrane proteins J. Am. Chem. Soc. 132, 5628– +
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22. 22
  Yeliseev, A. A., Wong, K. K., Soubias, O., and Gawrisch, K. (2005) Expression of human peripheral cannabinoid receptor for structural studies Protein Sci. 14, 2638– 2653
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  22
  Expression of human peripheral cannabinoid receptor for structural studies
  Yeliseev, Alexei A.; Wong, Karen K.; Soubias, Olivier; Gawrisch, Klaus
  Protein Science (2005), 14 (10), 2638-2653CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
  Human peripheral-type cannabinoid receptor (CB2) was expressed in Escherichia coli as a fusion with the maltose-binding protein, thioredoxin, and a deca-histidine tag. Functional activity and structural integrity of the receptor in bacterial protoplast membranes was confirmed by extensive binding studies with a variety of natural and synthetic cannabinoid ligands. E. coli membranes expressing CB2 also activated cognate G-proteins in an in vitro coupled assay. Detergent-solubilized receptor was purified to 80%-90% homogeneity by affinity chromatog. followed by ion-exchange chromatog. By high-resoln. NMR on the receptor in DPC micelles, it was detd. that purified CB2 forms 1:1 complexes with the ligands CP-55,940 and anadamide. The receptor was successfully reconstituted into phosphatidylcholine bilayers and the membranes were deposited into a porous substrate as tubular lipid bilayers for structural studies by NMR and scattering techniques.
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23. 23
  Oliver, R. C., Lipfert, J., Fox, D. A., Lo, R. H., Doniach, S., and Columbus, L. (2013) Dependence of micelle size and shape on detergent alkyl chain length and head group PLoS One 8, e62488
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  23
  Dependence of micelle size and shape on detergent alkyl chain length and head group
  Oliver, Ryan C.; Lipfert, Jan; Fox, Daniel A.; Lo, Ryan H.; Doniach, Sebastian; Columbus, Linda
  PLoS One (2013), 8 (5), e62488CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
  Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a sol. protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are detd. for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2-1.5 Å per carbon addn. to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approx. const. for each detergent series. In addn., the aggregation no. of these detergents increases by ∼16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.
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24. 24
  Van Horn, W. D., Kim, H. J., Ellis, C. D., Hadziselimovic, A., Sulistijo, E. S., Karra, M. D., Tian, C., Sonnichsen, F. D., and Sanders, C. R. (2009) Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase Science 324, 1726– 1729
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25. 25
  Verardi, R., Shi, L., Traaseth, N. J., Walsh, N., and Veglia, G. (2011) Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method Proc. Natl. Acad. Sci. U.S.A. 108, 9101– 9106
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26. 26
  Yao, Y., Bobkov, A. A., Plesniak, L. A., and Marassi, F. M. (2009) Mapping the interaction of pro-apoptotic tBID with pro-survival BCL-XL Biochemistry 48, 8704– 8711
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27. 27
  Chiu, M. L., Tsang, C., Grihalde, N., and MacWilliams, M. P. (2008) Over-expression, solubilization, and purification of G protein-coupled receptors for structural biology Comb. Chem. High Throughput Screening 11, 439– 462
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  27
  Over-expression, solubilization, and purification of G protein-coupled receptors for structural biology
  Chiu, Mark L.; Tsang, Cindy; Grihalde, Nelson; MacWilliams, Maria P.
  Combinatorial Chemistry & High Throughput Screening (2008), 11 (6), 439-462CODEN: CCHSFU; ISSN:1386-2073. (Bentham Science Publishers Ltd.)
  A review. With the advent of the recent detn. of high-resoln. crystal structures of bovine rhodopsin and human β2 adrenergic receptor (β2AR), there are still many structure-function relationships to be learned from other G protein-coupled receptors (GPCRs). Many of the pharmaceutically interesting GPCRs cannot be modeled because of their amino acid sequence divergence from bovine rhodopsin and β2AR. Structure detn. of GPCRs can provide new avenues for engineering drugs with greater potency and higher specificity. Several obstacles need to be overcome before membrane protein structural biol. becomes routine: over-expression, solubilization, and purifn. of milligram quantities of active and stable GPCRs. Coordinated iterative efforts are required to generate any significant GPCR over-expression. To formulate guidelines for GPCR purifn. efforts, we review published conditions for solubilization and purifn. using detergents and additives. A discussion of sample prepn. of GPCRs in detergent phase, bicelles, nanodiscs, or low-d. lipoproteins is presented in the context of potential structural biol. applications. In addn., a review of the solubilization and purifn. of successfully crystd. bovine rhodopsin and β2AR highlights tools that can be used for other GPCRs.
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28. 28
  Li, Q. X., Mittal, R., Huang, L. J., Travis, B., and Sanders, C. R. (2009) Bolaamphiphile-class surfactants can stabilize and support the function of solubilized integral membrane proteins Biochemistry 48, 11606– 11608
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29. 29
  Hanford, M. and Peeples, T. L. (2002) Archaeal tetraether lipids - unique structures and applications Appl. Biochem. Biotechnol. 97, 45– 62
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  Archaeal tetraether lipids: Unique structures and applications
  Hanford, Michael J.; Peeples, Tonya L.
  Applied Biochemistry and Biotechnology (2002), 97 (1), 45-62CODEN: ABIBDL; ISSN:0273-2289. (Humana Press Inc.)
  A review. The extremely stable biomols. manufd. by organisms from extreme environments are of great scientific and engineering interest in the development of robust and stable industrial biocatalysts. Identification of mols. that impart stability under extremes will also have a profound impact on our understanding of cellular survival. This review discusses isolation and characterization of archael tetraethers as well as target technologies for tetraether lipid application. The isolation and characterization of archaeal tetraether lipids has led to some interesting applications improving on ester lipid technologies. Potential applications include novel lubricants, gene-delivery systems, monolayer lipid matrixes for sensor devices, and protein stabilization. Following this review, patent abstrs. and addnl. literature pertaining to the isolation, characterization, and application of archaeal membrane lipids are listed.
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30. 30
  Ostuni, M. A., Iatmanen, S., Teboul, D., Robert, J. C., and Lacapere, J. J. (2010) Characterization of membrane protein preparations: measurement of detergent content and ligand binding after proteoliposomes reconstitution Methods Mol. Biol. 654, 3– 18
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  Characterization of membrane protein preparations: measurement of detergent content and ligand binding after proteoliposomes reconstitution
  Ostuni, Mariano A.; Iatmanen, Soria; Teboul, David; Robert, Jean-Claude; Lacapere, Jean-Jacques
  Methods in Molecular Biology (New York, NY, United States) (2010), 654 (Membrane Protein Structure Determination), 3-18CODEN: MMBIED; ISSN:1064-3745. (Springer)
  The study of membrane proteins is a difficult task due to their natural embedding in hydrophobic environment made by lipids. Solubilization and purifn. from native membranes or overexpressed system involves the use of detergent to make them sol. while maintaining their structural and functional properties. The choice of detergent is governed not only by their ability to reach these goals, but also by their compatibility with biochem. and structural studies. A different detergent can be used during purifn., and characterization of the detergent amts. present in each purifn. step is crucial. To address this point, we developed a colorimetric method to measure detergent content in different prepns. We analyzed detergent present in the collected fractions from the purifn. of the recombinant membrane translocator protein (RecTSPO). We followed detergent removal during the reconstitution of RecTSPO in liposomes and obsd. by electron microscopy the formation of proteoliposomes. We addressed the RecTSPO functionality by testing its ability to bind high affinity drug ligand [3H]PK 11195. We described the different parameters that should be controlled in order to optimize the measurement of this ligand binding using a filtration procedure. These protocols are useful to characterize functionality and detergent content of membrane protein, both key factors for further structural studies.
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31. 31
  Wang, L., Quan, C., Liu, B., Wang, J., Xiong, W., Zhao, P., and Fan, S. (2013) Functional reconstitution of staphylococcus aureus truncated agrc histidine kinase in a model membrane system PLoS One 8, e80400
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32. 32
  Zhou, H. X. and Cross, T. A. (2013) Influences of membrane mimetic environments on membrane protein structures Ann. Rev. Biophys. 42, 361– 392
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  Influences of membrane mimetic environments on membrane protein structures
  Zhou Huan-Xiang; Cross Timothy A
  Annual review of biophysics (2013), 42 (), 361-92 ISSN:.
  The number of membrane protein structures in the Protein Data Bank is becoming significant and growing. Here, the transmembrane domain structures of the helical membrane proteins are evaluated to assess the influences of the membrane mimetic environments. Toward this goal, many of the biophysical properties of membranes are discussed and contrasted with those of the membrane mimetics commonly used for structure determination. Although the mimetic environments can perturb the protein structures to an extent that potentially gives rise to misinterpretation of functional mechanisms, there are also many structures that have a native-like appearance. From this assessment, an initial set of guidelines is proposed for distinguishing native-like from nonnative-like membrane protein structures. With experimental techniques for validation and computational methods for refinement and quality assessment and enhancement, there are good prospects for achieving native-like structures for these very important proteins.
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33. 33
  Cross, T., Murray, D., and Watts, A. (2013) Helical membrane protein conformations and their environment Eur. Biophys. J. 42, 731– 755
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  Helical membrane protein conformations and their environment
  Cross, Timothy A.; Murray, Dylan T.; Watts, Anthony
  European Biophysics Journal (2013), 42 (10), 731-755CODEN: EBJOE8; ISSN:0175-7571. (Springer)
  A review. Evidence that membrane proteins respond conformationally and functionally to their environment is growing. Structural models, by necessity, have been characterized in prepns. where the protein has been removed from its native environment. Different structural methods have used various membrane mimetics that have recently included lipid bilayers as a more native-like environment. Structural tools applied to lipid bilayer-embedded integral proteins are informing us about important generic characteristics of how membrane proteins respond to the lipid environment as compared with their response to other nonlipid environments. Here, we review the current status of the field, with specific ref. to observations of some well-studied α-helical membrane proteins, as a starting point to aid the development of possible generic principles for model refinement.
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34. 34
  Zoonens, M., Comer, J., Masscheleyn, S., Pebay-Peyroula, E., Chipot, C., Miroux, B., and Dehez, F. (2013) Dangerous liaisons between detergents and membrane proteins. The case of mitochondrial uncoupling protein 2 J. Am. Chem. Soc. 135, 15174– 15182
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  White, J. F. and Grisshammer, R. (2010) Stability of the neurotensin receptor NTS1 free in detergent solution and immobilized to affinity resin PLoS One 5, e12579
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  Keller, M., Kerth, A., and Blume, A. (1997) Thermodynamics of interaction of octyl glucoside with phosphatidylcholine vesicles: partitioning and solubilization as studied by high sensitivity titration calorimetry Biochim. Biophys. Acta 1326, 178– 192
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  de la Maza, A. and Parra, J. L. (1997) Solubilizing effects caused by the nonionic surfactant dodecylmaltoside in phosphatidylcholine liposomes Biophys. J. 72, 1668– 1675
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  Tan, A., Ziegler, A., Steinbauer, B., and Seelig, J. (2002) Thermodynamics of sodium dodecyl sulfate partitioning into lipid membranes Biophys. J. 83, 1547– 1556
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  Thermodynamics of sodium dodecyl sulfate partitioning into lipid membranes
  Tan, Anmin; Ziegler, Andre; Steinbauer, Bernhard; Seelig, Joachim
  Biophysical Journal (2002), 83 (3), 1547-1556CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
  The partition equil. of sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate between water and bilayer membranes were investigated with isothermal titrn. calorimetry and spectroscopic methods (light scattering, 31P-NMR) in the temp. range of 28°C to 56°C. The partitioning of the dodecyl sulfate anion (DS-) into the bilayer membrane is energetically favored by an exothermic partition enthalpy of ΔHDo = -6.0 kcal/mol at 28°C. This is in contrast to nonionic detergents where ΔHDo is usually pos. The partition enthalpy decreases linearly with increasing temp. and the molar heat capacity is ΔCpo = -50 ± 3 cal mol-1 K-1. The partition isotherm is nonlinear if the bound detergent is plotted vs. the free detergent concn. in bulk soln. This is caused by the electrostatic repulsion between the DS- ions inserted into the membrane and those free in soln. near the membrane surface. The surface concn. of DS- immediately above the plane of binding was hence calcd. with the Gouy-Chapman theory, and a strictly linear relationship was obtained between the surface concn. and the extent of DS- partitioning. The surface partition const. K describes the chem. equil. in the absence of electrostatic effects. For the SDS-membrane equil. K was found to be 1.2 × 104 M-1 to 6 × 104 M-1 for the various systems and conditions investigated, very similar to data available for nonionic detergents of the same chain length. The membrane-micelle phase diagram was also studied. Complete membrane solubilization requires a ratio of 2.2 mol SDS bound per mol of total lipid at 56°C. The corresponding equil. concn. of SDS free in soln. is CD,fsol ∼ 1.7 mM and is slightly below the crit. micelles concn. (CMC) = 2.1 mM (at 56°C and 0.11 M buffer). Membrane satn. occurs at ∼ 0.3 mol SDS per mol lipid and the equil. SDS concn. is CD,fsat ≈ 2.2 mM ± 0.6 mM. SDS translocation across the bilayer is slow at ambient temp. but increases at high temps.
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39. 39
  Meister, A. and Blume, A. (2004) Solubilization of DMPC-d54 and DMPG-d54 vesicles with octylglucoside and sodium dodecyl sulfate studied by FT-IR spectroscopy Phys. Chem. Chem. Phys. 6, 1551– 1556
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  Cócera, M., López, O., Estelrich, J., Parra, J. L., and de la Maza, A. (2000) Kinetic and structural aspects of the adsorption of sodium dodecyl sulfate on phosphatidylcholine liposomes Langmuir 16, 4068– 4071
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  López, O., Cócera, M., Coderch, L., Parra, J. L., Barsukov, L., and de la Maza, A. (2001) Octyl glucoside-mediated solubilization and reconstitution of liposomes: structural and kinetic aspects J. Phys. Chem. B 105, 9879– 9886
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  Maza, A., Lopez, O., Baucells, J., Gonzalez, P., and Parra, J. L. (1998) Solubilization of phosphatidylcholine unilamellar liposomes caused by alkyl glucosides J. Surfactants Deterg. 1, 381– 386
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  Feng, G. and Winkler, M. E. (1995) Single-Step Purifications of His(6)-Muth, His(6)Mutl and His(6)-Muts Repair Proteins of Escherichia-Coli K-12 Biotechniques 19, 956– 965
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  Li, C. G., Gao, P., Qin, H. J., Chase, R., Gor’kov, P. L., Brey, W. W., and Cross, T. A. (2007) Uniformly aligned full-length membrane proteins in liquid crystalline bilayers for structural characterization J. Am. Chem. Soc. 129, 5304– 5305
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  Murray, D. T., Hung, I., and Cross, T. A. (2014) Assignment of oriented sample NMR resonances from a three transmembrane helix protein J. Magn. Reson. 240, 34– 44
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  Das, N., Murray, D. T., and Cross, T. A. (2013) Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples Nat. Protoc. 8, 2256– 2270
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  Nevzorov, A. A. and Opella, S. J. (2003) A “magic sandwich” pulse sequence with reduced offset dependence for high-resolution separated local field spectroscopy J. Magn. Reson. 164, 182– 186
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  Gor’kov, P. L., Chekmenev, E. Y., Li, C. G., Cotten, M., Buffy, J. J., Traaseth, N. J., Veglia, G., and Brey, W. W. (2007) Using low-E resonators to reduce RF heating in biological samples for static solid-state NMR up to 900 MHz J. Magn. Reson. 185, 77– 93
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  Takegoshi, K., Nakamura, S., and Terao, T. (2003) 13C–1H dipolar-driven 13C–13C recoupling without 13C rf irradiation in nuclear magnetic resonance of rotating solids J. Chem. Phys. 118, 2325– 2341
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  Takegoshi, K., Nakamura, S., and Terao, T. (2001) 13C–1H dipolar-assisted rotational resonance in magic-angle spinning NMR Chem. Phys. Lett. 344, 631– 637
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  McNeill, S. A., Gor’kov, P. L., Shetty, K., Brey, W. W., and Long, J. R. (2009) A low-E magic angle spinning probe for biological solid state NMR at 750 MHz J. Magn. Reson. 197, 135– 144
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  Fung, B. M., Khitrin, A. K., and Ermolaev, K. (2000) An improved broadband decoupling sequence for liquid crystals and solids J. Magn. Reson. 142, 97– 101
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  G protein-coupled receptors (GPCRs) have been found as monomers but also as dimers or higher-order oligomers in cells. The relevance of the monomeric or dimeric receptor state for G protein activation is currently under debate for class A rhodopsin-like GPCRs. Clarification of this issue requires the availability of well defined receptor prepns. as monomers or dimers and an assessment of their ligand-binding and G protein-coupling properties. We show by pharmacol. and hydrodynamic expts. that purified neurotensin receptor NTS1, a class A GPCR, dimerizes in detergent soln. in a concn.-dependent manner, with an apparent affinity in the low nanomolar range. At low receptor concns., NTS1 binds the agonist neurotensin with a Hill slope of ≈1; at higher receptor concns., neurotensin binding displays pos. cooperativity with a Hill slope of ≈2. NTS1 monomers activate Gαqβ1γ2, whereas receptor dimers catalyze nucleotide exchange with lower affinity. Our results demonstrate that NTS1 dimerization per se is not a prerequisite for G protein activation.
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Electron micrographs of the proteoliposome preparations obtained using SDS and DDGly mediated reconstitution. This material is available free of charge via the Internet at http://pubs.acs.org.
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Detergent Optimized Membrane Protein Reconstitution in Liposomes for Solid State NMR

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Abstract

Experimental Procedures

Protein Expression and Batch Purification

Size Exclusion Chromatography

CD Spectroscopy

Proteoliposome Preparation

Evaporative Light Scattering Detection

Oriented Sample Solid State NMR Samples and Spectroscopy

Magic Angle Spinning Solid State NMR Samples and Spectroscopy

Results

Detergent Choice

Figure 1

Batch Purification Assay

Figure 2

Oligomeric State in Micelles

Figure 3

Alpha Helical Content in Micelles

Figure 4

Detergent Removal

Figure 5

Solid State NMR Sample Quality

Figure 6

Figure 7

Discussion

Supporting Information

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Author Information

Acknowledgment

Abbreviations

References

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Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

References

Supporting Information

Supporting Information

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