Solution NMR studies of polytopic α-helical membrane proteins

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Solution NMR studies of polytopic a-helical membrane proteins Daniel Nietlispach and Antoine Gautier NMR spectroscopy has established itself as one of the main techniques for the structural study of integral membrane proteins. Remarkably, over the last few years, substantial progress has been achieved in the structure determination of increasingly complex polytopical a-helical membrane proteins, with their size approaching 100 kDa. Such advances are the result of significant improvements in NMR methodology, sample preparation and powerful selective isotope labelling schemes. We review the requirements facilitating such work based on the more recent solution NMR studies of a-helical proteins. While the majority of such studies still use detergent-solubilized proteins, alternative more native-like lipid-based media are emerging. Recent interaction, dynamics and conformational studies are discussed that cast a promising light on the future role of NMR in this important and exciting area. Address Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK Corresponding author: Nietlispach, Daniel ([email protected])

Current Opinion in Structural Biology 2011, 21:497–508 This review comes from a themed issue on Membranes Edited by Gebhard Franz Xaver Schertler and Robert Stroud Available online 19th July 2011 0959-440X/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2011.06.009

Introduction Integral membrane proteins (IMPs) are highly abundant in the genome, but structural information remains underrepresented. Currently, most IMP structures have been solved using X-ray crystallography but increasingly both solution and solid-state NMR spectroscopy are playing a more significant role and at present ca. 10–15% of the available membrane protein structures have been solved by NMR, a situation reminiscent of globular proteins. Of the two main membrane protein topology classes the ahelical IMPs have proved to be the most difficult to work with. This is mostly because of their limited stability, the multiple problems encountered during protein expression, their disposition to form less regular tertiary structures and the tendency of smaller a-helical proteins to assemble into oligomers. In contrast, b-barrel membrane proteins tend to have a more predictable topology and frequently are available in sufficient quantity through www.sciencedirect.com

over-expression into inclusion bodies in Escherichia coli (E. coli), followed by refolding into a functional form. One of the distinct advantages of NMR spectroscopy over other structure determination techniques is its ability to report on dynamical processes also. These are observable over a wide range of timescales and at atomic resolution. Thus, in some cases, NMR allows a comprehensive study of membrane protein structural as well as dynamical aspects. Alternatively, by reporting only on the dynamics, NMR can be used as a complementary method to other structural techniques, which provide only a static molecular picture. Interaction studies with ligands, membrane mimetics or other partner proteins can readily be monitored by following changes in the NMR spectra. Furthermore, binding affinities and kinetics can be determined and changes to global and local dynamics are immediately tractable. Over recent years, method developments, in both solution and solid-state NMR, have made comprehensive studies of larger proteins more accessible. Currently, solution methodology still seems to be more prominent and better suited to structural studies than solid-state NMR, but the two techniques are highly complementary and many of the methods used have progressively started to merge. Although substantial advances have been achieved in solid-state NMR [1,2], in this review we will focus primarily on the achievements using solution NMR. The increasingly frequent appearance of structures of larger membrane proteins solved by solution NMR is an indication that the methodology has now matured to a level where such studies can be tackled on a more routine basis. Major limitations still persist, mostly because of sample preparation, in particular the requirement for sufficient amounts of isotopically labelled proteins, choice of membrane mimetic, the need for long-term sample stability and an upper size limit for full structure determination of probably around 300–400 residues, depending upon the medium used for protein solubilization. This size limit is imposed by two features: the progressively broader NMR signals that ensue with increasing size of the detergentsolubilized protein and the detrimental effects of extensive isotope labelling required to obtain sufficient structural information. Dynamics and interaction studies on the other hand can be addressed in a more specific manner using selective labelling approaches. This leads to better NMR performance making such studies feasible well beyond the structural size limit. Guided by some of the more recent high-resolution NMR studies on larger monomeric and oligomeric, polytopic helical proteins (Figure 1), we assess the current situation and discuss the requirements to Current Opinion in Structural Biology 2011, 21:497–508

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Figure 1

DsbB

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Examples of recent a-helical structures determined by solution NMR. From left to right: The disulfide bond formation protein B DsbB [3] (PDB 2K73) with four TM-helices and sensory rhodopsin pSRII [4] (PDB 2KSY) with seven TM-helices are monomeric, while diacylglycerol kinase DAGK [6] (PDB 2KDC) is a domain-swapped trimer with three TM-helices per subunit. All three-protein structures were solved in detergent micelles. The potassium channel KcsA (PDB 2K1E, 2A9H) on the right is a tetramer of two TM-helices per subunit and is shown here as a water-soluble analogue WSK-3 [85].

enable NMR studies of IMP. We will also discuss future developments and their possible impact in this field.

Protein expression A fundamental limitation for NMR structural studies is the requirement of milligram quantities of isotopically labelled protein. Although many different expression methods exist, the heterologous preparation of samples in E. coli offers the benefits of larger yields and increased flexibility with isotope labelling. Sample preparation can be attempted through constitutive over-expression of the protein into the plasma membrane, followed by extraction from the membrane and transfer into detergent micelles as was demonstrated for the enzyme DsbB [3] and pSRII [4]. Alternatively, an approach employed for many b-barrel membrane proteins can be pursued where the protein is expressed into inclusion bodies. This approach requires refolding of the protein, as has been demonstrated for the M2 proton channel of influenza A virus [5] and DAGK [6]. Refolding of ahelical membrane proteins into a functional form often leads to very low yields. Encouragingly though, refolding with good yields was recently demonstrated for several class A G protein-coupled receptors (GPCRs) using amphipols [7,8]. As the suggested protocol seems general enough to be accessible to a wide range of a-helical proteins, expression into inclusion bodies could become a more viable strategy for the production of isotopically labelled material. Other prokaryotic and eukaryotic expression systems are available for the production of isotopically labelled membrane proteins [9]. Of these, expression in methylotrophic Pichia pastoris seems particularly promising. It allows the inclusion of certain post-translational modifications, results in the correct folding of proteins that contain multiple disulfide bonds and permits uniform 13 15 C, N-labelling [10] and even perdeuteration [11]. Current Opinion in Structural Biology 2011, 21:497–508

Selective isotope labelling in P. pastoris has been demonstrated but appears to be limited [12]. Recent progress with cell-free expression of a-helical transmembrane proteins makes this an interesting alternative to cellbased expression systems [13]. Uniform labelling is now commonly performed as well as perdeuteration [14] and amino acid selective labelling with low levels of scrambling has been achieved [15]. In the presence of detergents or lipids, substantial amounts of folded protein can be produced or alternatively the membrane protein can be expressed in insoluble form. Demonstrating the flexibility of the method, cell-free expression was recently used in combination with a sequence optimized combinatorial dual-labelling approach to achieve rapid backbone assignment for two a-helical two-transmembrane and a four-transmembrane histidine kinase receptor [16].

Membrane protein solubilization Solution NMR spectroscopy of a membrane protein requires solubilization of the latter using a membrane mimicking environment that can stabilize the protein over several days to record NMR spectra. At the same time the solubilized protein complex should not exceed a molecular size of 100 kDa should a full structural study be the intention. Currently, micelle-forming detergents remain the most popular medium, as they frequently result in the best quality NMR spectra. Many choices are available and although some candidates are more promising than others [9], a screening routine is typically required where the selected detergent, concentration and a broad range of conditions are optimized to provide the best NMR spectra. Detergents can create conditions for a protein that are quite different from the native membrane environment and the possible consequences of this need to be carefully assessed. To maintain the intrinsic properties of the solubilized membrane protein under study care needs to be taken when choosing a medium, and the www.sciencedirect.com

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functionality of the protein may need to be confirmed, for example by monitoring a ligand binding event. Depending upon the functionality test chosen, the interpretation can be convoluted, for example, a particular ligand may be found to bind but the interaction site may turn out to be of secondary importance. Using more realistic membranelike media such as small isotropic bicelles [17,18] or nanodiscs [19] are therefore beneficial and have already been successfully applied in a number of examples (reviewed in [20]). In solid-state NMR the absence of a direct correlation between molecular size and sample linewidths allows the use of bigger membrane media such as large bicelles or lipid bilayers, which provide a more native environment. Even so, in comparison to the complexity of real biological membranes, most membrane mimetics remain relatively coarse approximations. Biological membranes consist of highly complex lipid mixtures of varying compositions, which can adaptively adjust to change the

physical properties of the membrane. Lipid and protein concentration gradients are frequently found next to high densities of protein, and protein oligomerization can be mediated via specific interactions with lipids. Long-range organization and communication across the lipid bilayer have also been reported [21,22]. While such effects help to regulate the functional diversity of the proteins embedded within the membrane they are difficult to emulate in practice. Within their limits most membrane-mimetics employed today perform as relatively crude models. A meticulous optimization of detergent choice and sample conditions resulting in highly homogeneous and reproducible preparations for NMR is typically the key to the success of a project. In our experience even small variations in sample consistency can have detrimental effects on spectral appearance and NMR performance. Broad NMR signals may be the result of exchange broadening because of non-optimal sample conditions rather

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Preparation of highly homogeneous pSRII-micelle samples for NMR. 2D 1H–15N TROSY (transverse relaxation-optimized spectroscopy) spectra of pSRII (0.5 mM, pH 6) were recorded using varying amounts of c7-DHPC (di-heptanoyl-phosphatidylcholine). All DHPC concentrations (16.5–82.5 mM) are at least 10 above the CMC of the detergent. Increasing the DHPC concentration initially improves sample homogeneity and spectral quality until an optimum (ca. 110 excess) is reached. At even higher concentrations the NMR signals start to broaden as the increased viscosity leads to slower molecular reorientation. www.sciencedirect.com

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Effect of detergent choice on the spectral appearance of pSRII. Comparison of 2D 1H–15N TROSY spectra of sensory rhodopsin II solubilized in a range of micelle-forming detergents using u-15N pSRII: (a) LMPG (408C); (b) LOPG (408C); (c) LPPG (408C); (d) LMPC (508C); (e) c7-DHPC (408C) and u-2H,15N pSRII; (f) c7-DHPC (508C). All spectra were recorded at 800 MHz and detergent concentrations were individually optimized for best protein signal intensity (pSRII 0.4 mM, experiment times 3 h). Current Opinion in Structural Biology 2011, 21:497–508

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than reflecting an inherent dynamical property of the protein. For our work with pSRII [4], optimization of the solution conditions in di-heptanoyl-phosphatidylcholine (c7-DHPC) led from heterogeneously broadened peaks to high quality spectra, emphasizing the importance of high sample homogeneity (Figure 2). Once the conditions had been optimized, 1H,15N spectral dispersion and experimental performance were excellent. On the contrary, in suboptimal detergents, the spectral resolution was very low with the signals strongly broadened; resulting in very poorly dispersed and overlapped data (Figure 3).

Structure determination In parallel with the more frequent studies of larger bbarrel proteins, recent years have also seen the structure determination of increasingly larger and more complex ahelical systems such as KcsA [23], DsbB [3], DAGK [6] and pSRII [4] (Figure 1 and Table 1). Although the NMR spectra of symmetrical oligomers will benefit from peak redundancy, their larger molecular weight can make them very challenging systems to study, with large signal linewidths as encountered in the structure determinations of DAGK and KcsA. By comparison, monomeric proteins of an equivalent size have NMR spectra containing more peaks making them also very complex systems to analyse. In order to develop and test the methodology for the NMR structure determination of heptahelical membrane proteins recent work in our laboratory focused on the study of the microbial seven-helical receptor pSRII from Natronomonas pharaonis. Particular emphasis was placed on obtaining sufficient experimental restraints to provide high quality structural information of both the protein backbone and the side chain conformations (Figure 1).

A combination of high magnetic fields, TROSY spectroscopy and high levels of deuteration [24] in combination with maximum entropy processing [25] allowed the initial backbone assignment of the 241-residue pSRII in detergent micelles. Assignment of protein backbone resonances is often achievable up to a protein–detergent complex size of 100 kDa based on amide-directed backbone experiments and for helical proteins the use of sequential inter-amide NOESY (nuclear Overhauser effect spectroscopy) data allows an extension even beyond this limit. The use of deuterated detergents can further improve NMR performance and in particular the quality of NOESY spectra. With the backbone assignments of pSRII completed, the a-helical elements were delineated, based on chemical shift information and a combination of hydrogen bond restraints, inter-amide NOEs (nuclear Overhauser effects) and TALOS+ [26] derived backbone dihedral angle information, which helped to build an initial model of the seven individual a-helices. For the side chain assignment of pSRII a hybrid approach was chosen where initially the methyl containing residues were assigned using selectively ILV (isoleucine, leucine, valine) methyl-protonated samples [27]. This was followed by the use of protonated samples to obtain a larger number of inter-proton distances. Assignments were extended onto other non-methyl containing residues using 3D/4D NOESY and through bond correlation spectroscopy. 4D NOESY experiments recorded with nonuniform sampling and multi-dimensional decomposition (MDD) have been shown to reduce the substantial overlap that typically plagues the methyl region of 3D NOESY experiments [28]. Information derived from

Table 1 Selection of solution NMR studies of integral polytopic a-helical membrane proteins.a Protein (PDB ID)

Organism e

Expression system

Oligomeric state

Molecular weight

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Reference

pSRII (2KSY) DsbB (2K73) DAGK (2KDC) KdpP (2KSF) Phospholamban (1ZLL) M2 (2RLF) KcsA (2A9H) KcsA c KcsA c KcsAd (2KB1) TehA c Smr c Stt3p c

N. pharaonis E. coli E. coli E. coli H. sapiens Influenza virus S. lividans S. lividans S. lividans S. lividans E. coli S. aureus S. cerevisiae

E. coli E. coli E. coli Cell free E. coli E. coli E. coli E. coli E. coli E. coli Cell free E. coli E. coli

Monomer Monomer Trimer Monomer Pentamer Tetramer Tetramer Tetramer Tetramer Tetramer Monomer Dimer Monomer

26.7 kDa 20.9 kDa 39.3 kDa 11.4 kDa 30.5 kDa 20.7 kDa 71.1 kDa 68 kDa 71.1 kDa 45.6 kDa 24 kDa 23.4 kDa 31.5 kDa

c7-DHPC 508C DPC 408C DPC 458C LMPG 458C DPC 308C c6-DHPC 308C DPC 428C SDS 508C DPC 378C 208C LMPG 408C LPPG 478C SDS 558C

[4] [3] [6] [16] [86] [5] [23] [59] [60] [85] [87] [88] [89]

a

With four or more transmembrane helices. Abbreviations: c6-DHPC, di-hexanoyl-phosphatidylcholine; c7-DHPC, di-heptanoyl-phosphatidylcholine; DPC, dodecyl-phosphocholine; LMPG, 1-myristoyl-2-hydroxy-sn-glycero-3-[phosphor-rac-(1-glycerol)]; LPPG, 1-palmitoyl-2-hydroxy-sn-glycero-3-[phosphor-rac-(1-glycerol)]; SDS, sodium dodecyl sulfate. c Partial backbone resonance assignments. d Water-soluble analogue, no detergents were used. e Homo sapiens, Streptomyces lividans, Staphylococcus aureus, Saccharomyces cerevisiae. b

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alanines was found to be complementary to Ile, Leu and Val residues assisting with the assembly of the a-helices and their relative packing and orientation [4]. Methods that incorporate selectively methyl-protonated alanine in a highly deuterated background are therefore extremely valuable [29,30]. For pSRII the NOE-based approach resulted in more than 10 distance restraints per residue, many of which were long-range. As a result, the calculated structures showed extremely good packing with welldefined backbone and side chain conformations (Figure 1) approaching the quality of the crystal structures [31,32]. Selecting a side chain assignment strategy is determined by the size of the protein–detergent complex under study but also by the desired quality of the final structure ensemble. In the slow tumbling regime, characterized by increased signal linewidths and deteriorating quality of the NMR spectra, it may not be possible to obtain a sufficiently high number of NOEs from protonated samples to allow a satisfying description of the protein side chain orientations. In such cases, strictly selectiveprotonation based strategies should be more suitable, such as the cell-free expression geared Stereo-Array Isotope Labelling approach [33]. This method exploits spectroscopic improvements that come from the incorporation of amino acids with stereospecific position-selective as well as residue-selective protonation. Although very expensive, such a tailored approach can have various benefits and also offers a powerful alternative to provide restraints to the structurally important aromatic residues. Spin label derived paramagnetic relaxation enhancement (PRE) distances have been shown to be a particularly powerful way to obtain longer range information on larger proteins. Where NOE data are sparse, the addition of distance information derived from 7 to 10 MTSL (S(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate)) nitroxide labels introduced at single cysteine mutation sites can facilitate the tertiary assembly of an a-helical protein as was shown for DsbB [3], DAGK [6] and previously for several b-strand membrane proteins [34,35]. While PRE introduces an r 6 distance dependence, the use of paramagnetic metal tags can alternatively lead to pseudocontact shifts (PCS), which depend as r 3 on the electron-nucleus distance and the angle of the electronnucleus vector relative to a molecular frame. Like the commonly used MTSL nitroxide spin labels for PRE measurements, lanthanide metal tags can be attached to a surface cysteine via disulfide bond formation [36]. Recent work has seen the introduction of metal chelators that introduce more rigidity [37,38] and lead to larger PCS that can result in detectable effects up to 55 A˚ [39]. Their potential for membrane protein structure determination remains to be tested. Current Opinion in Structural Biology 2011, 21:497–508

Residual dipolar couplings have been successfully used for DAGK [6], DsbB [3], M2 [5] and other proteins [9] to improve the structural accuracy and precision. With the high concentrations of detergent normally used, polyacrylamide gel alignment media seem to be particularly well suited [9] and in the case of pSRII, a partial alignment could be achieved using collagen [40]. Efficient use of these orientational and complementary distance methods relies on the formation of the individual ahelices through sufficient NOEs between amide–amide, amide–methyl and methyl–methyl protons. Chemical shift based structure calculation methods have proven to be very successful on globular proteins [41–44] but have not yet been demonstrated on membrane proteins. At the moment these methods still rely on the availability of an extensive set of backbone and proton chemical shifts, which may be difficult to obtain for large solubilized membrane proteins. Very likely, improved algorithms that rely on less comprehensive chemical shift data sets will present a powerful way to provide structural information for membrane proteins.

Towards NMR structures of GPCRs Most of the a-helical membrane proteins studied so far are of microbial origin with only a few available eukaryotic examples. Not surprisingly therefore, the contribution of NMR to the structural understanding of GPCRs [45] as the largest class of a-helical proteins is still very limited when compared with the recent breakthrough achievements that have been obtained by X-ray crystallography over the last three years (reviewed in [46]). However, once solubilized, many of the class A GPCRs are not too dissimilar in size to KcsA, DAGK or pSRII, for which NMR structures have been determined. Many structural similarities with the latter microbial seventransmembrane receptor indicate that in principle, similar results should be achievable for GPCRs using comparable labelling techniques. While encouraging, the unavailability of such studies emphasizes where the real problems currently remain: limitations in the amounts of producible isotopically labelled material, low protein stability in nonmembrane environments, issues with sample homogeneity and the arduous process to study systems which are close to the current size limit for a full or even only partial backbone based global-fold determination. GPCRs are generally very unstable but thermal stabilization techniques have been pioneered [47], which do not lead to an increase in the overall size. They are also applicable to NMR work. Sample stabilities over several days can be achieved using thermostabilized mutants in order to facilitate longer NMR studies above ambient temperature [4]. The recent progress of X-ray based structure determination initially concentrated on the more stable antagonist www.sciencedirect.com

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and inverse agonist bound states of GPCRs [48] and has now also started to produce structures of agonist bound activated states [49–51]. This exciting trend is expected to develop further in the near future. Assuming that X-ray structures can be solved in sufficient numbers for different functional states then NMR-based approaches will be unable to compete with these rapid developments. Clearly, in the presence of hundreds of potential GPCR structures the temptation to pursue structure determination is very high and promises a substantial role for NMR in the future. At the same time, NMR techniques are better suited to the characterization of biophysical aspects such as the study of molecular (e.g. ligand) interactions with GPCRs or the study of transiently populated conformations and the analysis of protein dynamics on many timescales. In these situations, NMR allows to obtain unique mechanistic insight. NMR has the potential to report on conformational exchange processes that go along with activation and could convey structural features of lower populated, less stable GPCR states. Of particular interest are changes on the intracellular and extracellular surfaces that correlate with ligand binding [52]. Solution NMR studies with labelled GPCRs are relatively sparse to date and have concentrated on residue specific selectively labelled rhodopsin [53–55]. In all these studies, only a few NMR signals were observed from residues that reside in flexible regions of the protein while the helical transmembrane regions were in general not visible or strongly broadened. It is currently not clear if such broadening effects in the spectra are a particular of the rhodopsin dynamics, are characteristic of GPCRs in general, or are caused by the detrimental effects of residual sample heterogeneity given the detergents used. In our experience extreme sensitivity to the effects of sample heterogeneity was experienced with pSRII in several of the detergents tested.

Studying the membrane protein environment The observation that the structures of some membrane proteins may be substantially affected by the mimetic of the membrane bilayer has sparked a renewed interest in the relevance of the protein environment employed [5,56,57]. Assessing such influence on protein structure and function requires the characterization of interactions with lipids and detergents for which a wide range of methods are available. For example, monitoring the chemical shift changes during a titration showed a direct interaction of cholesterol with the C-terminal fragment of the amyloid precursor protein, indicating a possible connection between cholesterol homeostasis and Alzheimer’s disease [58]. Mapping of protein–lipid/detergent interactions via discrete NOEs was shown for the 68 kDa potassium channel KcsA [59,60], whereas cross-saturation methods were used to detect specific protein–lipid interactions [61]. Alternatively, paramagnetic broadening techniques using a combination of water-soluble and hydrophobic reagents www.sciencedirect.com

that reveal solvent accessible as well as buried hydrophobic sites have been used [4,62,63]. PCS reagents including transition metals and also dissolved oxygen at increased pressures were used for a similar purpose, with the latter reagent mapping the hydrophobic regions of the protein [64,65]. Large chemical shift changes which might require substantial reassignments can be elegantly circumvented by the selective introduction of 19F at known positions either as 19F-containing amino acids or as probes attached to cysteines [65]. Of particular benefit is the relative ease with which solidstate NMR is able to determine the orientation of helices relative to the membrane lipid bilayer normal [1,66]. The results of solid-state and solution NMR are complementary as has been shown for example with the single span FXYD family proteins [67]. Similarly, in an effort to combine the benefits of the two techniques, Veglia and co-workers discuss a hybrid approach using solution and solid-state NMR restraints to solve the structure of monomeric phospholamban in a lipid bilayer [68]. Supplementing their previous results in micelles, protein orientation, tilt and rotation relative to the lipid bilayer normal, depth of membrane insertion and the interaction of individual protein subunits with the lipid bilayer have now also been revealed [69]. This hybrid approach is suggested to provide structural and topological information for small membrane proteins where helix arrangements are strongly influenced through interactions with the native lipid bilayer. The possible influence of the detergent micelle moiety on protein function or structure has led to the use of improved more native-like membrane environments such as small bicelles and particles referred to as nanolipoproteins, nanodiscs or reconstituted high density lipoprotein [20]. As such, when small bicelle solubilized KCNE3, the modulatory protein of the K+ channel was injected into Xenopus oocytes, a functional co-assembly with human KCNQ1 was shown. In contrast, the micelle-solubilized protein did not lead to any channel modulation [70]. Stabilization of a small multidrug-resistance (Smr) protein in isotropic bicelles was shown together with the ability to obtain backbone assignment [18,71], whereas the function of Smr was strongly impaired in detergent environments. A similar effect was observed for the heterodimeric aIIb3 integrin which appeared in the associated off-state in bicelles, while no interaction was manifested in dodecyl-phosphocholine (DPC) micelles [72]. In contrast, Sanders and co-workers have shown that the catalytic activity of DAGK in lysophospholipid micelles is maintained in the absence of any lipids although they found substantial differences in DAGK–detergent interactions depending on the detergent used [73]. Nanodiscs provide a more native environment but with their size of 150 kDa can be challenging for more Current Opinion in Structural Biology 2011, 21:497–508

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comprehensive structural studies. The 1H,15N spectral appearance of a given protein in nanodiscs has been suggested as a reference point to find suitable, but smaller micellar detergents that create an environment similar to nanodiscs [74].

Ligand interaction studies, conformational changes and dynamics Several of the recent full structural studies of membrane proteins involved also the characterization of interactions with functionally relevant ligands. For example, the observation of intermolecular NOEs between DsbB and quinone allowed the location of the ligand at the periplasmic end of DsbB, in a different position compared to the crystal structure [3,75]. On the basis of NOEs, rimantadine binding to the influenza virus A proton channel M2 (18– 60) tetramer in DHPC was found to occur in a stoichiometric ratio to the outside of the channel [5]. Solid-state measurements on M2 (22–46) [56] conducted in a DMPC bilayer, on the other hand, located one amantadine per tetramer blocking the channel near the site of the S31N mutation related to drug resistance, in agreement with the crystal structure results. The solution NMR study was conducted in the presence of a large excess of the drug, which led to saturation of a lower affinity binding site, but no evidence of binding inside the channel itself was found. Again, these differences emphasize that a choice of membrane mimetic needs to be made very carefully and that the protein functionality needs to be cautiously assessed by additional methods. Over the years, prokaryotic KcsA has been studied by several NMR groups [23,59,60,76] producing a wealth of information on the dynamics and structural aspects that underlie the gating and permeation process of this tetrameric pH-dependent K+ channel. However, issues on how K+ conduction is switched on and off still remain elusive. A recent study performed on functional ILV methyl-protonated protein at low pH showed that residues located in the selectivity filter of the protein exchanged between two distinct conformations [77]. The spectral changes as a function of pH were mapped to the state of KcsA activation and further evidence of the conformational differences in the selectivity filter region were obtained from changes in NOE cross peaks. Combined with mutational evidence this provides prime structural evidence that the conformational changes in the selectivity filter of KcsA are coupled with the process of inactivation, that is, channel gating. Structural studies require milligram quantities of protein and are further limited by the upper size of the solubilized protein–detergent complexes. In contrast, for lower affinity interactions protein–protein and small ligand binding information can be obtained from dipolar interactions, chemical shift perturbation, differential spin relaxation and saturation transfer measurements. Current Opinion in Structural Biology 2011, 21:497–508

Ligands are generally small, giving rise to sharper signals. These are easier to observe allowing the determination of ligand conformations, interaction sites, binding constants and stoichiometries. With the difficulties that affect structural studies of GPCRs in particular, such investigations have been pursued for many years concentrating on ligand conformational aspects, receptor interactions and ligand screening in drug discovery [45,78]. Recently, the functional binding of brazzein to the heterodimeric Class C GPCR human sweet receptor in native membrane suspensions was shown using saturation transfer difference spectroscopy [79]. To observe a ligand with micromolar binding affinity, the required amount of protein is typically very low and with less than 100 mg, is easily in a range where similar studies with other receptors that express in low yields should become feasible. Using the sensitive methyl-directed transferred crosssaturation technique, Shimada and co-workers showed that the interaction of the detergent-solubilized class A chemokine receptor CXCR4 with the chemokine CXCL12/ SDF-1a involves two extended independent binding sites, in agreement with a two-step receptor–chemokine binding model [80]. They demonstrated that in a separate first binding event, the highly flexible N terminus of the chemokine is efficiently searching the binding pocket of the GPCR. The same group applied similar methyldirected transferred cross-saturation techniques to provide a structural analysis of the chemokine receptor CCR5 interaction with a monomeric macrophage inflammatory protein MIP-1a variant [81]. Structural studies of CCR5 have been hindered by low receptor stability in detergent micelles. Instead, they directly reconstituted the functional receptor from the Sf9 cell lysate into high-density lipoprotein. Using the lipids from the cell membrane the lifetime of CCR5 in nanodiscs could be extended to 24 hours, sufficiently long to map the interaction with the chemokine. As this promising method requires very small amounts of protein (1–10 mM) it should be well suited for other GPCR–ligand interaction studies involving low stability membrane proteins that require characterization in a lipid bilayer environment. Comparison of recent X-ray structures of the inactive b1adrenergic receptor (b1-AR), b2-AR, A2a receptors with rhodopsin has highlighted differences in the conformations of the second extracellular loops (EL2) which have been related to variations in their basal activity [82]. Probing the ligand-specific effects on EL2, Kobilka and co-workers selectively labelled the extracellular domain of the b2-AR with 13C via dimethylation of the lysine side chains to monitor ligand induced changes to the local environment of a salt bridge between Asp192 in EL2 and Lys305 in EL3 [83]. The two loops form part of the extracellular surface of b2-AR and the salt bridge hinders freely diffusible ligand access to the orthosteric www.sciencedirect.com

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binding site. Using the methyl group chemical shifts in 2D 1H–13C HMQC (heteronuclear multiple quantum correlation) spectra, the observed changes in the unliganded, inverse agonist and agonist bound states indicated a weakening of the salt bridge in the presence of the latter. Changes in 13C T2 relaxation times further implied restricted mobility of Lys305 in the salt bridge. Similarly, changes in a 2D TOCSY (total correlation spectroscopy) spectrum of the thrombosis and vasoconstriction mediating human thromboxane A2 GPCR provided evidence for an involvement of three out of a total of nine Trp residues in conformational changes upon agonist activation of the receptor [84]. It was speculated that two of the affected Trp residues were located in EL1 and EL2.

Conclusions Solution NMR studies of a-helical membrane proteins are progressing at a rapid pace and have contributed substantially towards the structure determination of numerous monomeric and oligomeric polytopic proteins, the characterization of protein interactions and the study of functionally related changes in conformation and dynamics. The majority of studies still use detergent-solubilized proteins but alternative and better-suited lipid-based media are emerging which allow functional studies in a more native environment. Viewed over the last few years, problems of increasing size and complexity have been addressed. The major facilitators of these developments involve the combined use of high magnetic field strengths, relaxation compensation NMR techniques, perdeuteration and diverse powerful and flexible selective isotope labelling strategies. Unsolved difficulties remain with the preparation of sufficient quantities of isotopically labelled proteins, particularly for the less stable eukaryotic proteins. Currently, the upper size limit of a system that can be structurally characterized using solution NMR methods approaches 150 kDa. To this point the high-resolution structure determination of a microbial seven-helical receptor and several other multi-helical proteins have been presented and this is casting a very optimistic outlook on the future contributions of solution NMR in the field of membrane protein studies.

Acknowledgement This work was supported by BBSRC grant BB/G011915/1.

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