Influence of the lipid membrane environment on structure and activity of the outer membrane protein Ail from Yersinia pestis

The surrounding environment has significant consequences for the structural and functional properties of membrane proteins. While native structure and function can be reconstituted in lipid bilayer membranes, the detergents used for protein solubilization are not always compatible with biological activity and, hence, not always appropriate for direct detection of ligand binding by NMR spectroscopy. Here we describe how the sample environment affects the activity of the outer membrane protein Ail (attachment invasion locus) from Yersinia pestis. Although Ail adopts the correct β-barrel fold in micelles, the high detergent concentrations required for NMR structural studies are not compatible with the ligand binding functionality of the protein. We also describe preparations of Ail embedded in phospholipid bilayer nanodiscs, optimized for NMR studies and ligand binding activity assays. Ail in nanodiscs is capable of binding its human ligand fibronectin and also yields high quality NMR spectra that reflect the proper fold. Binding activity assays, developed to be performed directly with the NMR samples, show that ligand binding involves the extracellular loops of Ail. The data show that even when detergent micelles support the protein fold, detergents can interfere with activity in subtle ways.     

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.     

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.     

How Amphipols Embed Membrane Proteins: Global Solvent Accessibility and Interaction with a Flexible Protein Terminus

Amphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded β-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins.     

Optimization of the design and preparation of nanoscale phospholipid bilayers for its application to solution NMR

Despite arduous efforts and recent technological developments structural investigation of integral membrane proteins remains a challenge. The primary deterrents include difficulties with their expression, low inherent solubility, and problems associated with existing membrane mimicking systems. A relatively new class of membrane mimetics, nanodiscs, is emerging as a promising alternative. Although nanodiscs have been proven successful for several biophysical applications, they yet remain to become the system of preferred choice for structure determination. We have hereby made nanodiscs more suitable for solution NMR applications by reducing the diameter of the self‐assembly complex to its potential limit. We achieved a noticeable improvement in the quality of NMR spectra obtained for the transmembrane and cytoplasmic domains of integrin α_IIb incorporated into these smaller discs rendering them susceptible for a thorough structural investigation. In addition, we also present an on‐column method for a rapid, efficient, single‐step preparation of protein incorporated nanodiscs at high concentrations. These discs have been fully characterized by transmission electron microscopy, dynamic light scattering, and differential scanning calorimetry. Proteins 2013; 81:1222–1231. © 2013 Wiley Periodicals, Inc.     

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.     

Characterization of protein–glycolipid recognition at the membrane bilayer

A growing number of important molecular recognition events are being shown to involve the interactions between proteins and glycolipids. Glycolipids are molecules in which one or more monosaccharides are glycosidically linked to a lipid moiety. The lipid moiety is generally buried in the cell membrane or other bilayer, leaving the oligosaccharide moiety exposed but in close proximity to the bilayer surface. This presents a unique environment for protein–carbohydrate interactions, and studies to determine the influence of the bilayer on these phenomena are in their infancy. One important property of the bilayer is the ability to orient and cluster glycolipid species, as strong interactions in biological systems are often achieved through multivalency arising from the simultaneous association of two or more proteins and receptors. This is especially true of protein–carbohydrate binding because of the unusually low affinities that characterize the monovalent interactions. More recent studies have also shown that the composition of the lipid bilayer is a critical parameter in protein–glycolipid recognition. The fluidity of the bilayer allows for correct geometric positioning of the oligosaccharide head group relative to the binding sites on the protein. In addition, there are activity‐based and structural data demonstrating the impact of the bilayer microenvironment on the modulation of oligosaccharide presentation. The use of model membranes in biosensor‐based methods has supplied decisive evidence of the importance of the membrane in receptor presentation. These data can be correlated with three‐dimensional structural information from X‐ray <?tw=98%>crystallography, NMR, and molecular mechanics to provide insight into specific protein–carbohydrate inter‐­actions at the bilayer. Copyright © 1999 National Research Council Canada and John Wiley & Sons, Ltd.     

NMR structure determination of a membrane protein with two transmembrane helices in micelles: MerF of the bacterial mercury detoxification system

The three-dimensional backbone structure of a membrane protein with two transmembrane helices in micelles was determined using solution NMR methods that rely on the measurement of backbone (1)H-(15)N residual dipolar couplings (RDCs) from samples of two different constructs that align differently in stressed polyacrylamide gels. Dipolar wave fitting to the (1)H-(15)N RDCs determines the helical boundaries based on periodicity and was utilized in the generation of supplemental dihedral restraints for the helical segments. The (1)H-(15)N RDCs and supplemental dihedral restraints enable the determination of the structure of the helix-loop-helix core domain of the mercury transport membrane protein MerF with a backbone RMSD of 0.58 A. Moreover, the fold of this polypeptide demonstrates that the two vicinal pairs of cysteine residues, shown to be involved in the transport of Hg(II) across the membrane, are exposed to the cytoplasm. This finding differs from earlier structural and mechanistic models that were based primarily on the somewhat atypical hydropathy plot for MerF and related transport proteins.     

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.     

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.     

Development of electron spin echo envelope modulation spectroscopy to probe the secondary structure of recombinant membrane proteins in a lipid bilayer

Membrane proteins conduct many important biological functions essential to the survival of organisms. However, due to their inherent hydrophobic nature, it is very difficult to obtain structural information on membrane‐bound proteins using traditional biophysical techniques. We are developing a new approach to probe the secondary structure of membrane proteins using the pulsed EPR technique of Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy. This method has been successfully applied to model peptides made synthetically. However, in order for this ESEEM technique to be widely applicable to larger membrane protein systems with no size limitations, protein samples with deuterated residues need to be prepared via protein expression methods. For the first time, this study shows that the ESEEM approach can be used to probe the local secondary structure of a ²H‐labeled d₈‐Val overexpressed membrane protein in a membrane mimetic environment. The membrane‐bound human KCNE1 protein was used with a known solution NMR structure to demonstrate the applicability of this methodology. Three different α‐helical regions of KCNE1 were probed: the extracellular domain (Val21), transmembrane domain (Val50), and cytoplasmic domain (Val95). These results indicated α‐helical structures in all three segments, consistent with the micelle structure of KCNE1. Furthermore, KCNE1 was incorporated into a lipid bilayer and the secondary structure of the transmembrane domain (Val50) was shown to be α‐helical in a more native‐like environment. This study extends the application of this ESEEM approach to much larger membrane protein systems that are difficult to study with X‐ray crystallography and/or NMR spectroscopy.     

Conformational transitions in the membrane scaffold protein of phospholipid bilayer nanodiscs

Phospholipid bilayer nanodiscs are model membrane systems that provide an environment where membrane proteins are highly stable and monodisperse without the use of detergents or liposomes. Nanodiscs consist of a discoidal phospholipid bilayer encircled by two copies of an amphipathic alpha helical membrane scaffold protein, which is modeled from apolipoprotein A-1. Hydrogen exchange mass spectrometry was used to probe the structure and dynamics of the scaffold protein in the presence and absence of lipid. On nanodisc self-assembly, the entire scaffold protein gained significant protection from exchange, consistent with a large, protein-wide, structural rearrangement. This protection was short-lived and the scaffold protein was highly deuterated within 2 h. Several regions of the scaffold protein, in both the lipid-free and lipid-associated states, displayed EX1 unfolding kinetics. The rapid deuteration of the scaffold protein and the presence of correlated unfolding events both indicate that nanodiscs are dynamic rather than rigid bodies in solution. This work provides a catalog of the expected scaffold protein peptic peptides in a nanodisc-hydrogen exchange mass spectrometry experiment and their deuterium uptake signatures, data that can be used as a benchmark to verify correct assembly and nanodisc structure. Such reference data will be useful control data for all hydrogen exchange mass spectrometry experiments involving nanodiscs in which transmembrane or lipid-associated proteins are the primary molecule(s) of interest.     

Nanodiscs versus macrodiscs for NMR of membrane proteins

It is challenging to find membrane mimics that stabilize the native structures, dynamics, and functions of membrane proteins. In a recent advance, nanodiscs have been shown to provide a bilayer environment compatible with solution NMR. We show that increasing the lipid to "belt" peptide ratio expands their diameter, slows their reorientation rate, and allows the protein-containing discs to be aligned in a magnetic field for oriented sample solid-state NMR. The spectroscopic properties of membrane proteins with one to seven transmembrane helices in q = 0.1 isotropic bicelles, ~10 nm diameter isotropic nanodiscs, ~30 nm diameter magnetically aligned macrodiscs, and q = 5 magnetically aligned bicelles are compared.     

Small‐angle X‐ray and neutron scattering demonstrates that cell‐free expression produces properly formed disc‐shaped nanolipoprotein particles

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native‐like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent‐solubilized protein‐lipid mixture. Recently, an alternative method has been developed using direct cell‐free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell‐free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell‐free methods for the production of lipid binding proteins. In addition, detailed structural information describing these NLPs can be obtained by fitting a capped core‐shell cylinder type model to all SANS/SAXS data simultaneously.     

Transmembrane helix-helix interactions: comparative simulations of the glycophorin a dimer

The glycophorin helix dimer is a paradigm for the exploration of helix-helix interactions in integral membrane proteins. Two NMR structures of the dimer are known, one in a detergent micelle and one in a lipid bilayer. Multiple (4 x 50 ns) molecular dynamics simulations starting from each of the two NMR structures, with each structure in either a dodecyl phosphocholine (DPC) micelle or a dimyristoyl phosphatidylcholine (DMPC) bilayer, have been used to explore the conformational dynamics of the helix dimer. Analysis of the helix-helix interaction, mediated by the GxxxG sequence motif, suggests convergence of the simulations to a common model. This is closer to the NMR structure determined in a bilayer than to micelle structure. The stable dimer interface in the final simulation model is characterized by (i) Gly/Gly packing and (ii) Thr/Thr interhelix H-bonds. These results demonstrate the ability of extended molecular dynamics simulations in a lipid bilayer environment to refine membrane protein structures or models derived from experimental data obtained in protein/detergent micelles.     

Escherichia coli diacylglycerol kinase: a case study in the application of solution NMR methods to an integral membrane protein

Diacylglycerol kinase (DAGK) is a 13-kDa integral membrane protein that spans the lipid bilayer three times and which is active in some micellar systems. In this work DAGK was purified using metal ion chelate chromatography, and its structural properties in micelles and organic solvent mixtures studies were examined, primarily to address the question of whether the structure of DAGK can be determined using solution NMR methods. Cross-linking studies established that DAGK is homotrimeric in decyl maltoside (DM) micelles and mixed micelles. The aggregate detergent-protein molecular mass of DAGK in both octyl glucoside and DM micelles was determined to be in the range of 100-110 kDa-much larger than the sum of the molecular weights of the DAGK trimers and the protein-free micelles. In acidic organic solvent mixtures, DAGK-DM complexes were highly soluble and yielded relatively well-resolved NMR spectra. NMR and circular dichroism studies indicated that in these mixtures the enzyme adopts a kinetically trapped monomeric structure in which it irreversibly binds several detergent molecules and is primarily alpha-helical, but in which its tertiary structure is largely disordered. Although these results provide new information regarding the native oligomeric state of DAGK and the structural properties of complex membrane proteins in micelles and organic solvent mixtures, the results discourage the notion that the structure of DAGK can be readily determined at high resolution with solution NMR methods.     

Membrane protein assembly into Nanodiscs

Nanodiscs are soluble nanoscale phospholipid bilayers which can self-assemble integral membrane proteins for biophysical, enzymatic or structural investigations. This means for rendering membrane proteins soluble at the single molecule level offers advantages over liposomes or detergent micelles in terms of size, stability, ability to add genetically modifiable features to the Nanodisc structure and ready access to both sides of the phospholipid bilayer domain. Thus the Nanodisc system provides a novel platform for understanding membrane protein function. We provide an overview of the Nanodisc approach and document through several examples many of the applications to the study of the structure and function of integral membrane proteins.     

Structure and dynamics of a membrane protein in micelles from three solution NMR experiments

Three solution NMR experiments on a uniformly ¹⁵N labeled membrane protein in micelles provide sufficient information to describe the structure, topology, and dynamics of its helices, as well as additional information that characterizes the principal features of residues in terminal and inter-helical loop regions. The backbone amide resonances are assigned with an HMQC-NOESY experiment and the backbone dynamics are characterized by a ¹H-¹⁵N heteronuclear NOE experiment, which clearly distinguishes between the structured helical residues and the more mobile residues in the terminal and interhelical loop regions of the protein. The structure and topology of the helices are described by Dipolar waves and PISA wheels derived from experimental measurements of residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs). The results show that the membrane-bound form of Pf1 coat protein has a 20-residue trans-membrane hydrophobic helix with an orientation that differs by about 90° from that of an 8-residue amphipathic helix. This combination of three-experiments that yields Dipolar waves and PISA wheels has the potential to contribute to high-throughput structural characterizations of membrane proteins.     

Characterization of the insertase BamA in three different membrane mimetics by solution NMR spectroscopy

The insertase BamA is the central protein of the Bam complex responsible for outer membrane protein biogenesis in Gram-negative bacteria. BamA features a 16-stranded transmembrane β-barrel and five periplasmic POTRA domains, with a total molecular weight of 88 kDa. Whereas the structure of BamA has recently been determined by X-ray crystallography, its functional mechanism is not well understood. This mechanism comprises the insertion of substrates from a dynamic, chaperone-bound state into the bacterial outer membrane, and NMR spectroscopy is thus a method of choice for its elucidation. Here, we report solution NMR studies of different BamA constructs in three different membrane mimetic systems: LDAO micelles, DMPC:DiC₇PC bicelles and MSP1D1:DMPC nanodiscs. The impact of biochemical parameters on the spectral quality was investigated, including the total protein concentration and the detergent:protein ratio. The barrel of BamA is folded in micelles, bicelles and nanodiscs, but the N-terminal POTRA5 domain is flexibly unfolded in the absence of POTRA4. Measurements of backbone dynamics show that the variable insertion region of BamA, located in the extracellular lid loop L6, features high local flexibility. Our work establishes biochemical preparation schemes for BamA, which will serve as a platform for structural and functional studies of BamA and its role within the Bam complex by solution NMR spectroscopy.     

Solution NMR spectroscopic characterization of human VDAC-2 in detergent micelles and lipid bilayer nanodiscs

Three isoforms of the human voltage-dependent anion channel (VDAC), located in the outer mitochondrial membrane, are crucial regulators of mitochondrial function. Numerous studies have been carried out to elucidate biochemical properties, as well as the three-dimensional structure of VDAC-1. However, functional and structural studies of VDAC-2 and VDAC-3 at atomic resolution are still scarce. VDAC-2 is highly similar to VDAC-1 in amino acid sequence, but has substantially different biochemical functions and expression profiles. Here, we report the reconstitution of functional VDAC-2 in lauryldimethylamine-oxide (LDAO) detergent micelles and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer nanodiscs. We find that VDAC-2 is properly folded in both membrane-mimicking systems and that structural and functional characterization by solution NMR spectroscopy is feasible. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.