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.     

Techniques and applications of NMR to membrane proteins (Review)

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.     

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.     

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.     

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.     

Novel chelate-induced magnetic alignment of biological membranes.

A phospholipid chelate complexed with ytterbium (DMPE-DTPA:Yb3+) is shown to be readily incorporated into a model membrane system, which may then be aligned in a magnetic field such that the average bilayer normal lies along the field. This so-called positively ordered smectic phase, whose lipids consist of less than 1% DMPE-DTPA:Yb3+, is ideally suited to structural studies of membrane proteins by solid-state NMR, low-angle diffraction, and spectroscopic techniques that require oriented samples. The chelate, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine diethylenetriaminepentaacetic acid, which strongly binds the lanthanide ions and serves to orient the membrane in a magnetic field, prevents direct lanthanide-protein interactions and significantly reduces paramagnetic shifts and line broadening. Similar low-spin lanthanide chelates may have applications in field-ordered solution NMR studies of water-soluble proteins and in the design of new magnetically aligned liquid crystalline phases.     

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.     

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.     

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.     

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.     

Solid-state NMR structure determination

Biological applications of solid-state NMR (SS-NMR) have been predominantly in the area of model membrane systems. Increasingly the focus has been membrane peptides and proteins. SS-NMR is able to provide information about how the peptides or proteins interact with the lipids or other peptides/proteins in the membrane, their effect on the membrane and the location of the peptides or proteins relative to the membrane surface. Recent developments in biological SS-NMR have been made possible by improvements in labelling and NMR techniques. This review discusses aligned systems and magic angle spinning techniques, bilayers and bicelles, and measurement of chemical shift anisotropy and dipolar coupling. A number of specific experiments such as cross polarization, rotational resonance, REDOR, PISEMA, MAOSS and multidimensional experiments are described. In addition to 2H, 13C and 15N, recent solid-sate 1H, 19F and 17O NMR work is discussed. Several examples of the use of SS-NMR to determine the structure of membrane peptides and proteins are given.     

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.     

Dynamic membrane interactions of antibacterial and antifungal biomolecules,and amyloid peptides,revealed by solid-state NMR spectroscopy

A variety of biomolecules acting on the cell membrane folds into a biologically active structure in the membrane environment. It is, therefore, important to determine the structures and dynamics of such biomolecules in a membrane environment. While several biophysical techniques are used to obtain low-resolution information, solid-state NMR spectroscopy is one of the most powerful means for determining the structure and dynamics of membrane bound biomolecules such as antibacterial biomolecules and amyloidogenic proteins; unlike X-ray crystallography and solution NMR spectroscopy, applications of solid-state NMR spectroscopy are not limited by non-crystalline, non-soluble nature or molecular size of membrane-associated biomolecules. This review article focuses on the applications of solid-state NMR techniques to study a few selected antibacterial and amyloid peptides. Solid-state NMR studies revealing the membrane inserted bent α-helical structure associated with the hemolytic activity of bee venom melittin and the chemical shift oscillation analysis used to determine the transmembrane structure (with α-helix and 3₁₀-helix in the N- and C-termini, respectively) of antibiotic peptide alamethicin are discussed in detail. Oligomerization of an amyloidogenic islet amyloid polypeptide (IAPP, or also known as amylin) resulting from its aggregation in a membrane environment, molecular interactions of the antifungal natural product amphotericin B with ergosterol in lipid bilayers, and the mechanism of lipid raft formation by sphingomyelin studied using solid state NMR methods are also discussed in this review article. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato.     

TROSY and CRINEPT: NMR with large molecular and supramolecular structures in solution

TROSY and CRINEPT are new techniques for solution NMR studies of molecular and supramolecular structures. They allow the collection of high-resolution spectra of structures with molecular weights >100 kDa, significantly extending the range of macromolecular systems that can be studied by NMR in solution. TROSY has already been used to map protein-protein interfaces, to conduct structural studies on membrane proteins and to study nucleic acid conformations in multimolecular assemblies. These techniques will help us to investigate the conformational states of individual macromolecular components and will support de novo protein structure determination in large supramolecular structures.     

Solid-state magic-angle spinning NMR of membrane proteins and protein-ligand interactions

Structural biology is developing into a universal tool for visualizing biological processes in space and time at atomic resolution. The field has been built by established methodology like X-ray crystallography, electron microscopy and solution NMR and is now incorporating new techniques, such as small-angle X-ray scattering, electron tomography, magic-angle-spinning solid-state NMR and femtosecond X-ray protein nanocrystallography. These new techniques all seek to investigate non-crystalline, native-like biological material. Solid-state NMR is a relatively young technique that has just proven its capabilities for de novo structure determination of model proteins. Further developments promise great potential for investigations on functional biological systems such as membrane-integrated receptors and channels, and macromolecular complexes attached to cytoskeletal proteins. Here, we review the development and applications of solid-state NMR from the first proof-of-principle investigations to mature structure determination projects, including membrane proteins. We describe the development of the methodology by looking at examples in detail and provide an outlook towards future 'big' projects.     

Solid-state NMR structures of integral membrane proteins

Abstract

Solid-state NMR is unique for its ability to obtain three-dimensional structures and to measure atomic-resolution structural and dynamic information for membrane proteins in native lipid bilayers. An increasing number and complexity of integral membrane protein structures have been determined by solid-state NMR using two main methods. Oriented sample solid-state NMR uses macroscopically aligned lipid bilayers to obtain orientational restraints that define secondary structure and global fold of embedded peptides and proteins and their orientation and topology in lipid bilayers. Magic angle spinning (MAS) solid-state NMR uses unoriented rapidly spinning samples to obtain distance and torsion angle restraints that define tertiary structure and helix packing arrangements. Details of all current protein structures are described, highlighting developments in experimental strategy and other technological advancements. Some structures originate from combining solid- and solution-state NMR information and some have used solid-state NMR to refine X-ray crystal structures. Solid-state NMR has also validated the structures of proteins determined in different membrane mimetics by solution-state NMR and X-ray crystallography and is therefore complementary to other structural biology techniques. By continuing efforts in identifying membrane protein targets and developing expression, isotope labelling and sample preparation strategies, probe technology, NMR experiments, calculation and modelling methods and combination with other techniques, it should be feasible to determine the structures of many more membrane proteins of biological and biomedical importance using solid-state NMR. This will provide three-dimensional structures and atomic-resolution structural information for characterising ligand and drug interactions, dynamics and molecular mechanisms of membrane proteins under physiological lipid bilayer conditions.     

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.     

The cytochromes P450 and b5 and their reductases--promising targets for structural studies by advanced solid-state NMR spectroscopy

Members of the cytochrome P450 (cyt P450) superfamily of enzymes oxidize a wide array of endogenous and xenobiotic substances to prepare them for excretion. Most of the drugs in use today are metabolized in part by a small set of human cyt P450 isozymes. Consequently, cyt P450s have for a long time received a lot of attention in biochemical and pharmacological research. Cytochrome P450 receives electrons from cytochrome P450 reductase and in selected cases from cytochrome b5 (cyt b5). Numerous structural studies of cyt P450s, cyt b5, and their reductases have given considerable insight into fundamental structure-function relationships. However, structural studies so far have had to rely on truncated variants of the enzymes to make conventional X-ray crystallographic and solution-state NMR techniques applicable. In spite of significant efforts it has not yet been possible to crystallize any of these proteins in their full-length membrane bound forms. The truncated parts of the enzymes are assumed to be alpha-helical membrane anchors that are essential for some key properties of cyt P450s. In the present contribution we set out with a basic overview on the current status of functional and structural studies. Our main aim is to demonstrate how advanced modern solid-state NMR spectroscopic techniques will be able to make substantial progress in cyt P450 research. Solid-state NMR spectroscopy has sufficiently matured over the last decade to be fully applicable to any membrane protein system. Recent years have seen a remarkable increase in studies on membrane protein structure using a host of solid-state NMR techniques. Solid-state NMR is the only technique available today for structural studies on full-length cyt P450 and full-length cyt b5. We aim to give a detailed account of modern techniques as applicable to cyt P450 and cyt b5, to show what has already been possible and what seems to be viable in the very near future.     

Use of reverse micelles in membrane protein structural biology

Membrane protein structural biology is a rapidly developing field with fundamental importance for elucidating key biological and biophysical processes including signal transduction, intercellular communication, and cellular transport. In addition to the intrinsic interest in this area of research, structural studies of membrane proteins have direct significance on the development of therapeutics that impact human health in diverse and important ways. In this article we demonstrate the potential of investigating the structure of membrane proteins using the reverse micelle forming surfactant dioctyl sulfosuccinate (AOT) in application to the prototypical model ion channel gramicidin A. Reverse micelles are surfactant based nanoparticles which have been employed to investigate fundamental physical properties of biomolecules. The results of this solution NMR based study indicate that the AOT reverse micelle system is capable of refolding and stabilizing relatively high concentrations of the native conformation of gramicidin A. Importantly, pulsed-field-gradient NMR diffusion and NOESY experiments reveal stable gramicidin A homodimer interactions that bridge reverse micelle particles. The spectroscopic benefit of reverse micelle-membrane protein solubilization is also explored, and significant enhancement over commonly used micelle based mimetic systems is demonstrated. These results establish the effectiveness of reverse micelle based studies of membrane proteins, and illustrate that membrane proteins solubilized by reverse micelles are compatible with high resolution solution NMR techniques. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.