Techniques and applications of NMR to membrane proteins

The fact that membrane proteins are notoriously difficult to analyse using standard protocols for atomic-resolution structure determination methods have motivated adaptation of these techniques to membrane protein studies as well as development of new technologies. With this motivation, liquid-state nuclear magnetic resonance (NMR) has recently been used with success for studies of peptides and membrane proteins in detergent micelles, and solid-state NMR has undergone a tremendous evolution towards characterization of membrane proteins in native membrane and oriented phospholipid bilayers. In this mini-review, we describe some of the technological challenges behind these efforts and provide examples on their use in membrane biology.     

NMR solution structure determination of membrane proteins reconstituted in detergent micelles

As an alternative to X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy in solution can be used for three-dimensional structure determination of small membrane proteins, preferably proteins with beta-barrel fold. This paper reviews recent achievements as well as limiting factors encountered in solution NMR studies of membrane proteins. Our particular interest has been focused on supplementing structure determination with data on the solvation of the proteins in the mixed micelles with detergents that are used to reconstitute membrane proteins for the NMR experiments. For the Escherichia coli outer membrane protein X (OmpX) in dihexanoylphosphatidylcholine (DHPC) micelles, such studies showed that the central part of the protein is covered with a fluid monolayer of lipid molecules, which seems to mimic quite faithfully the embedding of the protein in the lipid phase of the biological membrane. The implication is that the micellar systems used in this instance for the NMR studies of the membrane protein should also be suitable for further investigations of functional interactions with other proteins or low-molecular weight ligands.     

Solution NMR studies of the integral membrane proteins OmpX and OmpA from Escherichia coli

Membrane proteins are usually solubilized in polar solvents by incorporation into micelles. Even for small membrane proteins these mixed micelles have rather large molecular masses, typically beyond 50000 Da. The NMR technique TROSY (transverse relaxation-optimized spectroscopy) has been developed for studies of structures of this size in solution. In this paper, strategies for the use of TROSY-based NMR experiments with membrane proteins are discussed and illustrated with results obtained with the Escherichia coli integral membrane proteins OmpX and OmpA in mixed micelles with the detergent dihexanoylphosphatidylcholine (DHPC). For OmpX, complete sequence-specific NMR assignments have been obtained for the polypeptide backbone. The 13C chemical shifts and nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution, and in the collection of an input of conformational constraints for the computation of the global fold of the protein. For OmpA, the NMR assignments are so far limited to about 80% of the polypeptide chain, indicating different dynamic properties of the reconstituted OmpA beta-barrel from those of OmpX. Overall, the present data demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure, function and dynamics of integral membrane proteins.     

Solution NMR studies of polytopic α-helical membrane proteins

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 α-helical membrane proteins, with their size approaching ～100kDa. 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 α-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.     

Solution NMR of membrane proteins in bilayer mimics: small is beautiful, but sometimes bigger is better

Considerable progress has been made recently on solution NMR studies of multi-transmembrane helix membrane protein systems of increasing size. Careful correlation of structure with function has validated the physiological relevance of these studies in detergent micelles. However, larger micelle and bicelle systems are sometimes required to stabilize the active forms of dynamic membrane proteins, such as the bacterial small multidrug resistance transporters. Even in these systems with aggregate molecular weights well over 100 kDa, solution NMR structural studies are feasible-but challenging.     

Expression, purification, and characterization of Thermotoga maritima membrane proteins for structure determination

Structural studies of integral membrane proteins typically rely upon detergent micelles as faithful mimics of the native lipid bilayer. Therefore, membrane protein structure determination would be greatly facilitated by biophysical techniques that are capable of evaluating and assessing the fold and oligomeric state of these proteins solubilized in detergent micelles. In this study, an approach to the characterization of detergent-solubilized integral membrane proteins is presented. Eight Thermotoga maritima membrane proteins were screened for solubility in 11 detergents, and the resulting soluble protein-detergent complexes were characterized with small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and chemical cross-linking to evaluate the homogeneity, oligomeric state, radius of gyration, and overall fold. A new application of SAXS is presented, which does not require density matching, and NMR methods, typically used to evaluate soluble proteins, are successfully applied to detergent-solubilized membrane proteins. Although detergents with longer alkyl chains solubilized the most proteins, further characterization indicates that some of these protein-detergent complexes are not well suited for NMR structure determination due to conformational exchange and protein oligomerization. These results emphasize the need to screen several different detergents and to characterize the protein-detergent complex in order to pursue structural studies. Finally, the physical characterization of the protein-detergent complexes indicates optimal solution conditions for further structural studies for three of the eight overexpressed membrane proteins.     

Solution NMR of membrane proteins in bilayer mimics: Small is beautiful, but sometimes bigger is better

Considerable progress has been made recently on solution NMR studies of multi-transmembrane helix membrane protein systems of increasing size. Careful correlation of structure with function has validated the physiological relevance of these studies in detergent micelles. However, larger micelle and bicelle systems are sometimes required to stabilize the active forms of dynamic membrane proteins, such as the bacterial small multidrug resistance transporters. Even in these systems with aggregate molecular weights well over 100 kDa, solution NMR structural studies are feasible—but challenging.     

Structure determination of membrane proteins in five easy pieces

Rotational Alignment (RA) solid-state NMR provides the basis for a general method for determining the structures of membrane proteins in phospholipid bilayers under physiological conditions. Membrane proteins are high priority targets for structure determination, and are challenging for existing experimental methods. Because membrane proteins reside in liquid crystalline phospholipid bilayer membranes it is important to study them in this type of environment. The RA solid-state NMR approach we have developed can be summarized in five steps, and incorporates methods of molecular biology, biochemistry, sample preparation, the implementation of NMR experiments, and structure calculations. It relies on solid-state NMR spectroscopy to obtain high-resolution spectra and residue-specific structural restraints for membrane proteins that undergo rotational diffusion around the membrane normal, but whose mobility is otherwise restricted by interactions with the membrane phospholipids. High resolution spectra of membrane proteins alone and in complex with other proteins and ligands set the stage for structure determination and functional studies of these proteins in their native, functional environment.     

Customizing model membranes and samples for NMR spectroscopic studies of complex membrane proteins

Both solution and solid state nuclear magnetic resonance (NMR) techniques for structural determination are advancing rapidly such that it is possible to contemplate bringing these techniques to bear upon integral membrane proteins having multiple transmembrane segments. This review outlines existing and emerging options for model membrane media for use in such studies and surveys the special considerations which must be taken into account when preparing larger membrane proteins for NMR spectroscopic studies.     

NMR structures of polytopic integral membrane proteins

Membrane proteins represent up to 30% of the proteins in all organisms, they are involved in many biological processes and are the molecular targets for around 50% of validated drugs. Despite this, membrane proteins represent less than 1% of all high-resolution protein structures due to various challenges associated with applying the main biophysical techniques used for protein structure determination. Recent years have seen an explosion in the number of high-resolution structures of membrane proteins determined by NMR spectroscopy, especially for those with multiple transmembrane-spanning segments. This is a review of the structures of polytopic integral membrane proteins determined by NMR spectroscopy up to the end of the year 2010, which includes both β-barrel and α-helical proteins from a number of different organisms and with a range in types of function. It also considers the challenges associated with performing structural studies by NMR spectroscopy on membrane proteins and how some of these have been overcome, along with its exciting potential for contributing new knowledge about the molecular mechanisms of membrane proteins, their roles in human disease, and for assisting drug design.     

Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra

One of the biggest challenges in solid-state NMR studies of membrane proteins is to obtain a homogeneous natively folded sample giving high spectral resolution sufficient for structural studies. Eukaryotic membrane proteins are especially difficult and expensive targets in this respect. Methylotrophic yeast Pichia pastoris is a reliable producer of eukaryotic membrane proteins for crystallography and a promising economical source of isotopically labeled proteins for NMR. We show that eukaryotic membrane protein human aquaporin 1 can be doubly (¹³C/¹⁵N) isotopically labeled in this system and functionally reconstituted into phospholipids, giving excellent resolution of solid-state magic angle spinning NMR spectra.     

NMR structures of polytopic integral membrane proteins

Abstract

Membrane proteins represent up to 30% of the proteins in all organisms, they are involved in many biological processes and are the molecular targets for around 50% of validated drugs. Despite this, membrane proteins represent less than 1% of all high-resolution protein structures due to various challenges associated with applying the main biophysical techniques used for protein structure determination. Recent years have seen an explosion in the number of high-resolution structures of membrane proteins determined by NMR spectroscopy, especially for those with multiple transmembrane-spanning segments. This is a review of the structures of polytopic integral membrane proteins determined by NMR spectroscopy up to the end of the year 2010, which includes both β-barrel and α-helical proteins from a number of different organisms and with a range in types of function. It also considers the challenges associated with performing structural studies by NMR spectroscopy on membrane proteins and how some of these have been overcome, along with its exciting potential for contributing new knowledge about the molecular mechanisms of membrane proteins, their roles in human disease, and for assisting drug design.     

Investigation of the utility of selective methyl protonation for determination of membrane protein structures

Polytopic alpha-helical membrane proteins present one of the final frontiers for protein structural biology, with significant challenges causing severe under-representation in the protein structure databank. However, with the advent of hardware and methodology geared to the study of large molecular weight complexes, solution NMR is being increasingly considered as a tool for structural studies of these types of membrane proteins. One method that has the potential to facilitate these studies utilizes uniformly deuterated samples with protons reintroduced at one or two methyl groups of leucine, valine and isoleucine. In this work we demonstrate that in spite of the increased proportion of these amino acids in membrane proteins, the quality of structures that can be obtained from this strategy is similar to that obtained for all alpha-helical water soluble proteins. This is partly attributed to the observation that NOEs between residues within the transmembrane helix did not have an impact on structure quality. Instead the most important factors controlling structure accuracy were the strength of dihedral angle restraints imposed and the number of unique inter-helical pairs of residues constrained by NOEs. Overall these results suggest that the most accurate structures will arise from accurate identification of helical segments and utilization of inter-helical distance restraints from various sources to maximize the distribution of long-range restraints.     

The Advanced Properties of Circularized MSP Nanodiscs Facilitate High-resolution NMR Studies of Membrane Proteins

Membrane mimetics are essential for structural and functional studies of membrane proteins. A promising lipid-based system are phospholipid nanodiscs, where two copies of a so-called membrane scaffold protein (MSP) wrap around a patch of lipid bilayer. Consequently, the size of a nanodisc is determined by the length of the MSP. Furthermore, covalent MSP circularization was reported to improve nanodisc stability. However, a more detailed comparative analysis of the biophysical properties of circularized and linear MSP nanodiscs for their use in high-resolution NMR has not been conducted so far. Here, we analyze the membrane fluidity and temperature-dependent size variability of circularized and linear nanodiscs using a large set of analytical methods. We show that MSP circularization does not alter the membrane fluidity in nanodiscs. Further, we show that the phase transition temperature increases for circularized versions, while the cooperativity decreases. We demonstrate that circularized nanodiscs keep a constant size over a large temperature range, in contrast to their linear MSP counterparts. Due to this size stability, circularized nanodiscs are beneficial for high-resolution NMR studies of membrane proteins at elevated temperatures. Despite their slightly larger size as compared to linear nanodiscs, 3D NMR experiments of the voltage-dependent anion channel 1 (VDAC1) in circularized nanodiscs have a markedly improved spectral quality in comparison to VDAC1 incorporated into linear nanodiscs of a similar size. This study provides evidence that circularized MSP nanodiscs are a promising tool to facilitate high-resolution NMR studies of larger and challenging membrane proteins in a native lipid environment.     

On the use of <Emphasis Type="Italic">Pichia pastoris</Emphasis> for isotopic labeling of human GPCRs for NMR studies

NMR studies of human integral membrane proteins provide unique opportunities to probe structure and dynamics at specific locations and on multiple timescales, often with significant implications for disease mechanism and drug development. Since membrane proteins such as G protein-coupled receptors (GPCRs) are highly dynamic and regulated by ligands or other perturbations, NMR methods are potentially well suited to answer basic functional questions (such as addressing the biophysical basis of ligand efficacy) as well as guiding applications (such as novel ligand design). However, such studies on eukaryotic membrane proteins have often been limited by the inability to incorporate optimal isotopic labels for NMR methods developed for large protein/lipid complexes, including methyl TROSY. We review the different expression systems for production of isotopically labeled membrane proteins and highlight the use of the yeast Pichia pastoris to achieve perdeuteration and ¹³C methyl probe incorporation within isoleucine sidechains. We further illustrate the use of this method for labeling of several biomedically significant GPCRs.     

Membrane Protein Structural Validation by Oriented Sample Solid-State NMR: Diacylglycerol Kinase

The validation of protein structures through functional assays has been the norm for many years. Functional assays perform this validation for water-soluble proteins very well, but they need to be performed in the same environment as that used for the structural analysis. This is difficult for membrane proteins that are often structurally characterized in detergent environments, although functional assays for these proteins are most frequently performed in lipid bilayers. Because the structure of membrane proteins is known to be sensitive to the membrane mimetic environment, such functional assays are appropriate for validating the protein construct, but not the membrane protein structure. Here, we compare oriented sample solid-state NMR spectral data of diacylglycerol kinase previously published with predictions of such data from recent structures of this protein. A solution NMR structure of diacylglycerol kinase has been obtained in detergent micelles and three crystal structures have been obtained in a monoolein cubic phase. All of the structures are trimeric with each monomer having three transmembrane and one amphipathic helices. However, the solution NMR structure shows typical perturbations induced by a micelle environment that is reflected in the predicted solid-state NMR resonances from the structural coordinates. The crystal structures show few such perturbations, especially for the wild-type structure and especially for the monomers that do not have significant crystal contacts. For these monomers the predicted and observed data are nearly identical. The thermostabilized constructs do show more perturbations, especially the A41C mutation that introduces a hydrophilic residue into what would be the middle of the lipid bilayer inducing additional hydrogen bonding between trimers. These results demonstrate a general technique for validating membrane protein structures with minimal data obtained from membrane proteins in liquid crystalline lipid bilayers by oriented sample solid-state NMR.     

Solid-state NMR spectroscopy to study protein–lipid interactions

The appropriate lipid environment is crucial for the proper function of membrane proteins. There is a tremendous variety of lipid molecules in the membrane and so far it is often unclear which component of the lipid matrix is essential for the function of a respective protein. Lipid molecules and proteins mutually influence each other; parameters such as acyl chain order, membrane thickness, membrane elasticity, permeability, lipid-domain and annulus formation are strongly modulated by proteins. More recent data also indicates that the influence of proteins goes beyond a single annulus of next-neighbor boundary lipids. Therefore, a mesoscopic approach to membrane lipid–protein interactions in terms of elastic membrane deformations has been developed. Solid-state NMR has greatly contributed to the understanding of lipid–protein interactions and the modern view of biological membranes. Methods that detect the influence of proteins on the membrane as well as direct lipid–protein interactions have been developed and are reviewed here. Examples for solid-state NMR studies on the interaction of Ras proteins, the antimicrobial peptide protegrin-1, the G protein-coupled receptor rhodopsin, and the K⁺ channel KcsA are discussed. This article is part of a Special Issue entitled Tools to study lipid functions.     

Structural biology of transmembrane domains: efficient production and characterization of transmembrane peptides by NMR

Structural characterization of transmembrane peptides (TMPs) is justified because transmembrane domains of membrane proteins appear to often function independently of the rest of the protein. However, the challenge in obtaining milligrams of isotopically labeled TMPs to study these highly hydrophobic peptides by nuclear magnetic resonance (NMR) is significant. In the present work, a protocol is developed to produce, isotopically label, and purify TMPs in high yield as well as to initially characterize the TMPs with CD and both solution and solid-state NMR. Six TMPs from three integral membrane proteins, CorA, M2, and KdpF, were studied. CorA and KdpF are from Mycobacterium tuberculosis, while M2 is from influenza A virus. Several milligrams of each of these TMPs ranging from 25 to 89 residues were obtained per liter of M9 culture. The initial structural characterization results showed that these peptides were well folded in both detergent micelles and lipid bilayer preparations. The high yield, the simplicity of purification, and the convenient protocol represents a suitable approach for NMR studies and a starting point for characterizing the transmembrane domains of membrane proteins.     

The impact of window functions on NMR-based paramagnetic relaxation enhancement measurements in membrane proteins

Though challenging, solution NMR spectroscopy allows fundamental interrogation of the structure and dynamics of membrane proteins. One major technical hurdle in studies of helical membrane proteins by NMR is the difficulty of obtaining sufficient long range NOEs to determine tertiary structure. For this reason, long range distance information is sometimes sought through measurement of paramagnetic relaxation enhancements (PRE) of NMR nuclei as a function of distance from an introduced paramagnetic probe. Current PRE interpretation is based on the assumption of Lorentzian resonance lineshapes. However, in order to optimize spectral resolution, modern multidimensional NMR spectra are almost always subjected to resolution-enhancement, leading to distortions in the Lorentizian peak shape. Here it is shown that when PREs are derived using peak intensities (i.e., peak height) and linewidths from both real and simulated spectra that were produced using a wide range of apodization/window functions, that there is little variation in the distances determined (< 1 Å at the extremes). This indicates that the high degree of resolution enhancement required to obtain well-resolved spectra from helical membrane proteins is compatible with the use of PRE data as a source of distance restraints. While these conclusions are particularly important for helical membrane proteins, they are generally applicable to all PRE measurements made using resolution-enhanced data.     

Choosing membrane mimetics for NMR structural studies of transmembrane proteins

The native environment of membrane proteins is complex and scientists have felt the need to simplify it to reduce the number of varying parameters. However, experimental problems can also arise from oversimplification which contributes to why membrane proteins are under-represented in the protein structure databank and why they were difficult to study by nuclear magnetic resonance (NMR) spectroscopy. Technological progress now allows dealing with more complex models and, in the context of NMR studies, an incredibly large number of membrane mimetics options are available. This review provides a guide to the selection of the appropriate model membrane system for membrane protein study by NMR, depending on the protein and on the type of information that is looked for. Beside bilayers (of various shapes, sizes and lamellarity), bicelles (aligned or isotropic) and detergent micelles, this review will also describe the most recent membrane mimetics such as amphipols, nanodiscs and reverse micelles. Solution and solid-state NMR will be covered as well as more exotic techniques such as DNP and MAOSS.