Disulfide-Containing Detergents (DCDs) for the Structural Biology of Membrane Proteins

Disulfide-containing detergents (DCDs) are introduced, which contain a disulfide bond in the hydrophobic tail. DCDs form smaller micelles than corresponding detergents with linear hydrocarbon chains, while providing good solubilization and reconstitution of membrane proteins. The use of this new class of detergents in structural biology is illustrated with solution NMR spectra of the human G protein-coupled receptor A_2AAR, which is an α-helical protein, and the β-barrel protein OmpX from E. coli.     

Sequence-specific resonance assignment of soluble nonglobular proteins by 7D APSY-NMR spectroscopy

Based on sequence-specific resonance assignments, NMR is the method of choice for obtaining atomic-resolution experimental data on soluble nonglobular proteins. So far, however, NMR assignment of unfolded polypeptides in solution has been a time-consuming task, mainly due to the small chemical shift dispersion, which has limited practical applications of the NMR approach. This paper presents an efficient, fully automated method for sequence-specific backbone and beta-carbon NMR assignment of soluble nonglobular proteins with sizes up to at least 150 residues. The procedure is based on new APSY (automated projection spectroscopy) experiments which benefit from the short effective rotational correlation times in soluble nonglobular polypeptides to record five- to seven-dimensional NMR data sets, which reliably resolves chemical shift degeneracies. Fully automated sequence-specific resonance assignments of the backbone nuclei and C(beta) are described for the uniformly (13)C,(15)N-labeled urea-denatured 148-residue outer membrane protein X (OmpX) from E. coli. The method is generally applicable to systems with similar spectroscopic properties as unfolded OmpX, and we anticipate that this paper may open the door for extensive atomic-resolution studies of chemical denaturant-unfolded proteins, as well as some classes of functional nonglobular polypeptides in solution.     

Micelles,Bicelles, and Nanodiscs: Comparing the Impact of Membrane Mimetics on Membrane Protein Backbone Dynamics

Detergents are often used to investigate the structure and dynamics of membrane proteins. Whereas the structural integrity seems to be preserved in detergents for many membrane proteins, their functional activity is frequently compromised, but can be restored in a lipid environment. Herein we show with per‐residue resolution that while OmpX forms a stable β‐barrel in DPC detergent micelles, DHPC/DMPC bicelles, and DMPC nanodiscs, the pico‐ to nanosecond and micro‐ to millisecond motions differ substantially between the detergent and lipid environment. In particular for the β‐strands, there is pronounced dynamic variability in the lipid environment, which appears to be suppressed in micelles. This unexpected complex and membrane‐mimetic‐dependent dynamic behavior indicates that the frequent loss of membrane protein activity in detergents might be related to reduced internal dynamics and that membrane protein activity correlates with lipid flexibility.     

Monitoring Backbone Hydrogen‐Bond Formation in β‐Barrel Membrane Protein Folding

β‐barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three‐dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β‐strands. Here, we employ hydrogen–deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue‐specific kinetics of interstrand hydrogen‐bond formation were found to be uniform in the entire β‐barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long‐lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate‐limiting transition state and thus appears cooperative on the overall folding time scale.     

Translational diffusion of macromolecular assemblies measured using transverse-relaxation-optimized pulsed field gradient NMR

In structural biology, pulsed field gradient (PFG) NMR spectroscopy for the characterization of size and hydrodynamic parameters of macromolecular solutes has the advantage over other techniques that the measurements can be recorded with identical solution conditions as used for NMR structure determination or for crystallization trials. This paper describes two transverse-relaxation-optimized (TRO) (15)N-filtered PFG stimulated-echo (STE) experiments for studies of macromolecular translational diffusion in solution, (1)H-TRO-STE and (15)N-TRO-STE, which include CRINEPT and TROSY elements. Measurements with mixed micelles of the Escherichia coli outer membrane protein X (OmpX) and the detergent Fos-10 were used for a systematic comparison of (1)H-TRO-STE and (15)N-TRO-STE with conventional (15)N-filtered STE experimental schemes. The results provide an extended platform for evaluating the NMR experiments available for diffusion measurements in structural biology projects involving molecular particles with different size ranges. An initial application of the (15)N-TRO-STE experiment with very long diffusion delays showed that the tedradecamer structure of the 800 kDa Thermus thermophilus chaperonin GroEL is preserved in aqueous solution over the temperature range 25-60 °C.     

Bilayer in small bicelles revealed by lipid-protein interactions using NMR spectroscopy

Intermolecular nuclear Overhauser effects (NOEs) between the integral outer membrane protein OmpX from Escherichia coli and small bicelles of dihexanoyl phosphatidylcholine (DHPC) and dimyristoyl phosphatidylcholine (DMPC) give insights into protein-lipid interactions. Intermolecular NOEs between hydrophobic tails of lipid and protein in the bicelles cover the surface area of OmpX forming a continuous cylindric jacket of approximately 2.7 nm in height. These NOEs originate only from DMPC molecules, and no NOEs from DHPC are observed. Further, these NOEs are mainly from methylene groups of the hydrophobic tails of DMPC, and only a handful of NOEs arise from methyl groups of the hydrophobic tails. The observed contacts indicate that the hydrophobic tails of DMPC are oriented parallel to the surface of OmpX and thus DMPC molecules form a bilayer in the vicinity of the protein. Thus, a bilayer exists in the small bicelles not only in the absence of but also in the presence of a membrane protein. In addition, the number of NOEs between the polar head groups of lipid molecules and protein is increased in the bicelles compared with those in micelles. This observation may be due to the closely packed head groups of the bilayer. Moreover, irregularity of hydrophobic interactions in the middle of the bilayer environment was observed. This observation together with the interactions between polar head groups and proteins gives a possible rationale for structural and functional differences of membrane proteins solubilized in micelles and in bilayer systems and hints at structural differences between protein-free and protein-loaded bilayers.     

Separation of peptides from detergents using ion mobility spectrometry

Mass spectrometry (MS) has dramatically evolved in the last two decades and has been the driving force of the spectacular expansion of proteomics during this period. However, the very poor compatibility of MS with detergents is still a technical obstacle in some studies, in particular on membrane proteins. Indeed, the high hydrophobicity of membrane proteins necessitates the use of detergents for their extraction and solubilization. Here, we address the analytical potential of high-field asymmetric waveform ion mobility spectrometry (FAIMS) for separating peptides from detergents. The study was focused on peptides from the human integral membrane protein CD9. A tryptic peptide was mixed with the non-ionic detergents Triton X-100 or beta-D-dodecyl maltoside (DDM) as well as with the ionic detergents sodium dodecyl sulfate (SDS) or sodium deoxycholate (SDC). Although electrospray ionization (ESI) alone led to a total suppression of the peptide ion signal on mass spectra with only detection of the detergents, use of FAIMS allowed separation and clear identification of the peptide with any of the detergents studied. The detection and identification of the target compound in the presence of an excess of detergents are then feasible. FAIMS should prove especially useful in the structural and proteomic analysis of membrane proteins.     

Mesitylene‐Cored Glucoside Amphiphiles (MGAs) for Membrane Protein Studies: Importance of Alkyl Chain Density in Detergent Efficacy

Detergents serve as useful tools for membrane protein structural and functional studies. Their amphipathic nature allows detergents to associate with the hydrophobic regions of membrane proteins whilst maintaining the proteins in aqueous solution. However, widely used conventional detergents are limited in their ability to maintain the structural integrity of membrane proteins and thus there are major efforts underway to develop novel agents with improved properties. We prepared mesitylene‐cored glucoside amphiphiles (MGAs) with three alkyl chains and compared these agents with previously developed xylene‐linked maltoside agents (XMAs) with two alkyl chains and a conventional detergent (DDM). When these agents were evaluated for four membrane proteins including a G protein‐coupled receptor (GPCR), some agents such as MGA‐C13 and MGA‐C14 resulted in markedly enhanced stability of membrane proteins compared to both DDM and the XMAs. This favourable behaviour is due likely to the increased hydrophobic density provided by the extra alkyl chain. Thus, this study not only describes new glucoside agents with potential for membrane protein research, but also introduces a new detergent design principle for future development.     

Anesthetic modulation of protein dynamics: insight from an NMR study

Mistic (membrane integrating sequence for translation of integral membrane protein constructs) comprises the four-alpha-helix bundle scaffold found in the transmembrane domains of the Cys-loop receptors that are plausible targets for general anesthetics. Nuclear magnetic resonance (NMR) studies of anesthetic halothane interaction with Mistic in dodecyl phosphocholine (DPC) micelles provide an experimental basis for understanding molecular mechanisms of general anesthesia. Halothane was found to interact directly with Mistic, mostly in the interfacial loop regions. Although the presence of halothane had little effect on Mistic structure, (15)N NMR relaxation dispersion measurements revealed that halothane affected Mistic's motion on the microsecond-millisecond time scale. Halothane shifted the equilibrium of chemical exchange in some residues and made the exchange faster or slower in comparison to the original state in the absence of halothane. The motion on the microsecond-millisecond time scale in several residues disappeared in response to the addition of halothane. Most of the residues experiencing halothane-induced dynamics changes also exhibited profound halothane-induced changes in chemical shift, suggesting that dynamics modification of these residues might result from their direct interaction with halothane molecules. Allosteric modulation by halothane also contributed to dynamics changes, as reflected in residues I52 and Y82 where halothane introduction brought about dynamics changes but not chemical shift changes. The study suggests that inhaled general anesthetics could act on proteins via altering protein motion on the microsecond-millisecond time scale, especially motion in the flexible loops that link different alpha helices. The validation of anesthetic effect on protein dynamics that are potentially correlated with protein functions is a critical step in unraveling the mechanisms of anesthetic action on proteins.     

Bead-linked proteoliposomes: a reconstitution method for nmr analyses of membrane protein-ligand interactions

Structural information about the interactions between membrane proteins and their ligands provides insights into the membrane protein functions. A variety of surfactants have been used for structural analyses of membrane proteins, and in some cases, they yielded successful results. However, the use of surfactants frequently increases the conformational instability of membrane proteins and distorts their normal function. Here, we propose a new strategy of membrane protein reconstitution into lipid bilayers on affinity beads, which maintains the native conformation and function of the protein for NMR studies. The reconstituted membrane proteins are suitable for NMR analyses of interactions, by using the transferred cross-saturation method. The strategy was successfully applied to the interaction between a potassium ion channel, KcsA, and a pore-blocker, agitoxin2 (AgTx2). This strategy would be useful for analyzing the interactions between various membrane proteins and their ligands.     

Structural Studies of Self‐Assembled Subviral Particles: Combining Cell‐Free Expression with 110 kHz MAS NMR Spectroscopy

Viral membrane proteins are prime targets in combatting infection. Still, the determination of their structure remains a challenge, both with respect to sample preparation and the need for structural methods allowing for analysis in a native‐like lipid environment. Cell‐free protein synthesis and solid‐state NMR spectroscopy are promising approaches in this context, the former with respect to its great potential in the native expression of complex proteins, and the latter for the analysis of membrane proteins in lipids. Herein, we show that milligram amounts of the small envelope protein of the duck hepatitis B virus (DHBV) can be produced by cell‐free expression, and that the protein self‐assembles into subviral particles. Proton‐detected 2D NMR spectra recorded at a magic‐angle‐spinning frequency of 110 kHz on <500 μg protein show a number of isolated peaks with line widths comparable to those of model membrane proteins, paving the way for structural studies of this protein that is homologous to a potential drug target in HBV infection.     

Self-Assembly Behavior and Application of Terphenyl-Cored Trimaltosides for Membrane-Protein Studies: Impact of Detergent Hydrophobic Group Geometry on Protein Stability

Amphipathic agents are widely used in various fields including biomedical sciences. Micelle-forming detergents are particularly useful for in vitro membrane-protein characterization. As many conventional detergents are limited in their ability to stabilize membrane proteins, it is necessary to develop novel detergents to facilitate membrane-protein research. In the current study, we developed novel trimaltoside detergents with an alkyl pendant-bearing terphenyl unit as a hydrophobic group, designated terphenyl-cored maltosides (TPMs). We found that the geometry of the detergent hydrophobic group substantially impacts detergent self-assembly behavior, as well as detergent efficacy for membrane-protein stabilization. TPM-Vs, with a bent terphenyl group, were superior to the linear counterparts (TPM-Ls) at stabilizing multiple membrane proteins. The favorable protein stabilization efficacy of these bent TPMs is likely associated with a binding mode with membrane proteins distinct from conventional detergents and facial amphiphiles. When compared to n-dodecyl-β-d -maltoside (DDM), most TPMs were superior or comparable to this gold standard detergent at stabilizing membrane proteins. Notably, TPM-L3 was particularly effective at stabilizing the human β₂ adrenergic receptor (β₂AR), a G-protein coupled receptor, and its complex with G_s protein. Thus, the current study not only provides novel detergent tools that are useful for membrane-protein study, but also suggests a critical role for detergent hydrophobic group geometry in governing detergent efficacy.     

Solid-State NMR of a Large Membrane Protein by Paramagnetic Relaxation Enhancement

Membrane proteins play an important role in many biological functions. Solid-state NMR spectroscopy is uniquely suited for studying structure and dynamics of membrane proteins in a membranous environment. The major challenge to obtain high quality solid-state NMR spectra of membrane proteins is sensitivity, due to limited quantities of labeled high-molecular-weight proteins. Here we demonstrate the incorporation of paramagnetic metal (Cu(2+)) ions, through either EDTA or a chelator lipid, into membrane protein samples for rapid data collection under fast magic-angle spinning (MAS) and low power (1)H decoupling. Spectral sensitivity of DsbB (20 kDa), an integral membrane protein, more than doubles in the same experimental time due to (1)H T(1) relaxation enhancement by Cu(2+) ions, with DsbB native fold and active site intact. This technique can be implemented to acquire multidimensional solid-state NMR spectra for chemical shift assignments and structure elucidation of large membrane proteins with small sample quantities.     

Isolation of a Potential Anchoring Motif Based on Proteome Analysis of Escherichia coli and Its Use for Cell Surface Display

For bacterial cell surface display, the target protein needs to be linked to an anchoring motif, and it is essential to choose an appropriate anchoring motif for efficient and stable display of the protein on the cell surface. To isolate a potential anchoring motif that would allow a stable and enhanced display of target proteins on the surface of an Escherichia coli host, we analyzed the outer membrane proteome of E. coli. On the basis of this proteomic analysis, the outer membrane protein X (OmpX), which has a small, monomeric β-barrel structure and is highly expressed, was selected as a potential anchoring motif. The role of OmpX as an anchoring motif for cell surface display was demonstrated using three important industrial enzymes: endoxylanase, lipase, and alkaline phosphatase. Two different positions (Lys¹²², Val¹⁶⁰) in the extracellular loops of OmpX were examined for C-terminal fusion, and the biological activities and localization of the displayed enzymes were analyzed. All three enzymes examined were efficiently displayed on the E. coli cell surface with high activity. These results reveal that the use of OmpX as an anchoring motif is an efficient method to display functional enzymes on the surface of an E. coli host.     

Structure, topology, and dynamics of membrane peptides and proteins from solid-state NMR spectroscopy

The high-resolution structure of membrane proteins is notoriously difficult to determine due to the hydrophobic nature of the protein-membrane complexes. Solid-state NMR spectroscopy is a unique and powerful atomic-resolution probe of the structure and dynamics of these important biological molecules. A number of new solid-state NMR methods for determining the depth of insertion, orientation, oligomeric structure, and long-range (10-15 A) distances of membrane proteins are summarized. Membrane protein depths can now be determined using several complementary techniques with varying site-specificity, distance precision, and mobility requirement on the protein. Membrane protein orientation can now be determined with or without macroscopic alignment, the latter providing a novel alternative for orientation determination of intrinsically curvature-inducing proteins. The novel analyses of beta-sheet membrane protein orientation are described. The quaternary structure of membrane peptide assemblies can now be elucidated using a 19F spin diffusion technique that simultaneously yields the oligomeric number and intermolecular distances up to 15 A. Finally, long-range distances up to approximately 10 A can now be measured using 1H spins with an accuracy of better than 1 A. These methods are demonstrated on several beta-sheet membrane peptides with antimicrobial activities and on two alpha-helical ion-channel proteins. Finally, we show that the nearly ubiquitous dynamics of membrane proteins can be readily examined using 2D correlation experiments. An intimate appreciation of molecular motion in these systems not only leads to important insights into the specific function of these membrane proteins but also may be exploited for other purposes such as orientation determination.     

Development of 1,3-acetonedicarboxylate-derived glucoside amphiphiles (ACAs) for membrane protein study

Detergents are extensively used for membrane protein manipulation. Membrane proteins solubilized in conventional detergents are prone to denaturation and aggregation, rendering downstream characterization of these bio-macromolecules difficult. Although many amphiphiles have been developed to overcome the limited efficacy of conventional detergents for protein stabilization, only a handful of novel detergents have so far proved useful for membrane protein structural studies. Here, we introduce 1,3-acetonedicarboxylate-derived amphiphiles (ACAs) containing three glucose units and two alkyl chains as head and tail groups, respectively. The ACAs incorporate two different patterns of alkyl chain attachment to the core detergent unit, generating two sets of amphiphiles: ACA-As (asymmetrically alkylated) and ACA-Ss (symmetrically alkylated). The difference in the attachment pattern of the detergent alkyl chains resulted in minor variation in detergent properties such as micelle size, critical micelle concentration, and detergent behaviors toward membrane protein extraction and stabilization. In contrast, the impact of the detergent alkyl chain length on protein stability was marked. The two C11 variants (ACA-AC11 and ACA-SC11) were most effective at stabilizing the tested membrane proteins. The current study not only introduces new glucosides as tools for membrane protein study, but also provides detergent structure–property relationships important for future design of novel amphiphiles.

Newly developed amphiphiles, designated ACAs, are not only efficient at extracting G protein-coupled receptors from the membranes, but also conferred enhanced stability to the receptors compared to the gold standards (DDM and LMNG).     

Towards a native environment: structure and function of membrane proteins in lipid bilayers by NMR

Solid-state NMR (ssNMR) is a versatile technique that can be used for the characterization of various materials, ranging from small molecules to biological samples, including membrane proteins. ssNMR can probe both the structure and dynamics of membrane proteins, revealing protein function in a near-native lipid bilayer environment. The main limitation of the method is spectral resolution and sensitivity, however recent developments in ssNMR hardware, including the commercialization of 28 T magnets (1.2 GHz proton frequency) and ultrafast MAS spinning (<100 kHz) promise to accelerate acquisition, while reducing sample requirement, both of which are critical to membrane protein studies. Here, we review recent advances in ssNMR methodology used for structure determination of membrane proteins in native and mimetic environments, as well as the study of protein functions such as protein dynamics, and interactions with ligands, lipids and cholesterol.

Solid-state NMR (ssNMR) is a versatile technique that can be used for the characterization of various materials, ranging from small molecules to biological samples, including membrane proteins, as reviewed here.     

Isolation of an outer membrane protein complex from Borrelia burgdorferi by n-butanol extraction and high-performance ion-exchange chromatography

Borrelia burgdorferi, the causative agent of Lyme disease, expresses two major membrane proteins, designated outer surface proteins A and B, which are of antigenic relevance, especially in the chronic phase of Lyme disease. Both proteins exhibit strain-related molecular weight variation. A method is described for obtaining these proteins from the bacterial membrane, without the use of detergents, by a combination of n-butanol extraction and cation-exchange chromatography on a Mono S fast protein liquid chromatographic column. This method yields up to five times larger amounts of the proteins in aqueous solution than previously described protocols, which applied ionic or non-ionic detergents. A comparison of extracts obtained by this method from different Borrelia burgdorferi strains is reported.     

Membrane alignment of the pore-forming component TatA(d) of the twin-arginine translocase from Bacillus subtilis resolved by solid-state NMR spectroscopy

The twin-arginine translocase (Tat) provides protein export in bacteria and plant chloroplasts and is capable of transporting fully folded proteins across the membrane. We resolved the conformation and membrane alignment of the pore-forming subunit TatA(d) from Bacillus subtilis using solid-state NMR spectroscopy. The relevant structured part of the protein, TatA(2-45), contains a transmembrane segment (TMS) and an amphiphilic helix (APH). It was reconstituted in planar bicelles, which represent the lipid environment of a bacterial membrane. The SAMMY solid-state NMR experiment was used to correlate (15)N chemical shifts and (1)H-(15)N dipolar couplings in the backbone and side chains of the (15)N-labeled protein. The observed wheel-like patterns ("PISA wheels") in the resulting 2-dimensional spectra confirm the α-helical character of the two segments and reveal their alignment in the lipid bilayer. Helix tilt angles (τ(TMS) = 13°, τ(APH) = 64°) were obtained from uniformly labeled protein, and azimuthal rotations (ρ(Val15) = 235°, ρ(Ile29) = 25°) were obtained from selective labels. These constraints define two distinct families of allowed structures for TatA in the membrane-bound state. The manifold of solutions could be narrowed down to a unique structure by using input from a liquid-state NMR study of TatA in detergent micelles, as recently described [Hu, Y.; Zhao, E.; Li, H.; Xia, B.; Jin, C. J. Am. Chem. Soc. 2010, DOI: 10.1021/ja1053785]. Interestingly, the APH showed an unexpectedly slanted alignment in the protein, different from that of the isolated APH peptide. This finding implies that the amphiphilic region of TatA is not just a flexible attachment to the transmembrane anchor but might be able to form intra- or even intermolecular salt-bridges, which could play a key role in pore assembly.