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.     

Secondary structure, phospholipid membrane interactions, and fusion activity of two glutamate-rich analogs of influenza hemagglutinin fusion peptide

Two synthetic mutants of influenza HA2 fusion peptide (residues 1-25), containing Glu on the polar (residues 4,8-E5(4,8)) or the hydrophobic (residues 3,7-E5(3,7)) face of the amphipathic helix, were synthesized and labeled with NBD at the N-terminus. Introduction of Glu residues into the fusion peptide leads to increased sensitivity of various biochemical properties to pH compared to the wild type. The E5 peptides showed a decrease of alpha-helix content and increase of beta-sheet structure. Lipid binding was diminished, but not abolished even at high pH. The E5 analogs penetrate the lipid bilayer less deeply than the wild type, especially at high pH. The N-terminal half of the peptide showed significant variation of the depth of the penetration into the lipid bilayer. Both E5 peptides were fusion active. The properties of E5(3,7) were more affected by the Glu substitution and showed greater variation with pH than E5(4,8).     

Fast-time scale dynamics of outer membrane protein A by extended model-free analysis of NMR relaxation data

In order to better understand the dynamics of an integral membrane protein, backbone amide ¹⁵N NMR dynamics measurements of the β-barrel membrane protein OmpA have been performed at three magnetic fields. A total of nine relaxation data sets were globally analyzed using an extended model-free formalism. The diffusion tensor was found to be prolate axially symmetric with an axial ratio of 5.75, indicating a possible rotation of the protein within the micelle. The generalized order parameters gradually decreased from the mid-plane towards the two ends of the barrel, counteracting the dynamic gradient of the lipids in a matching bilayer, and were dramatically reduced in the extracellular loops. Large-scale internal motions on the ns time scale indicate that entire loops most likely undergo concerted (“sea anemone”-like) motions emanating from their anchoring points on the barrel. The case of OmpA in DPC micelles also illustrates inherent limitations of analyzing the data with even the most sophisticated current models of the model-free formalism. It is likely that conformational exchange processes on the ms-μs also play a role in describing the motions of some residues, but their analysis did not produce unique results that could be independently verified.     

Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12

The 325-residue outer membrane protein OmpA of Escherichia coli has been proposed to consist of a membrane-embedded moiety (residues 1 to about 170) and a C-terminal periplasmic region. The former is thought to comprise eight transmembrane segments in the form of antiparallel -strands, forming an amphiphilic connected by exposed turns. Several questions concerning this model were addressed. Thus no experimental evidence had been presented for the turns at the inner leaflet of the membrane and it was not known whether or not the periplasmic part of the polypeptide plays a role in the process of membrane incorporation. Oligonucleotides encoding trypsin cleavage sites were inserted at the predicted turn sites of the ompA gene and it was shown that the encoded proteins indeed become accessible to trypsin at the modified sites. Together with previous results, these data also show that the turns on both sides of the membrane do not possess specifically topogenic information. In two cases one of the two expected tryptic fragments was lost and could be detected at low concentration in only one case. Therefore, bilateral proteolytic digestion of outer membranes can cause loss of -strands and does not necessarily produce a reliable picture of protein topology. When ompA genes were constructed coding for proteins ending at residue 228 or 274, the membrane assembly of these proteins was shown to be partially defective with about 20% of the proteins not being assembled. No such defect was observed when, following the introduction of a premature stop codon, a truncated protein was produced ending with residue 171. It is concluded that (1) the proposed -barrel structure is essentially correct and (2) the periplasmic part of OmpA does not play an active role in, but can, when present in mutant form, interfere with membrane assembly.     

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.     

Molecular dynamics simulations of the influenza hemagglutinin fusion peptide in micelles and bilayers: conformational analysis of peptide and lipids

Molecular dynamics simulations of the influenza hemagglutinin fusion peptide in two differently sized dodecylphosphocholine micelles and a palmitoyl oleoyl phosphatidylcholine bilayer were generated to analyze the influence of the environment. Four independent trajectories (5 ns each for the bilayer, and 2 ns each for the micelles) were generated for each system. The peptide lies at the surface of the micelles, while its N-terminal region inserts deeply in the bilayer. This leads to a substantial increase of the solvation and rigidity of the peptide in micelles as compared to the bilayer. The average structures, nevertheless, are similar in all three systems and agree reasonably with micelle-based NMR structures. When in the bilayer, the peptide increases the chain gauche population and area of adjacent lipids in the same binding leaflet, while it has the opposite effect for the nearby lipids of the other leaflet. These changes, which occur spontaneously to fill voids and defects, cause a decrease in the thickness of the membrane in the neighborhood of the peptide. They would be expected to promote positive curvature, as consistent with the formation of the convex bulge, or "nipple", in the initial stage of membrane fusion. An extension of the classical surfactant theory of Israelachvili based on shapes is proposed to introduce the concept of a "dynamically induced shape" of the membrane lipids by the peptide.     

NMR assignments of the sylvatic dengue 1 virus envelope protein domain III

Nearly complete backbone and side chain resonance assignments have been obtained for the third domain, residues M289–K400, of the envelope protein from the sylvatic strain (P72–1244) of the dengue 1 virus, containing mutations N336S and E370K, using double- and triple-resonance spectroscopy.     

Recombinant outer membrane protein A (OmpA) of Edwardsiella tarda, a potential vaccine candidate for fish, common carp

Outer membrane protein A (OmpA) is a component of the outer membrane of Edwardsiella tarda and is wildly distributed in Enterobacteriaceae family. The gene encoding the OmpA protein was cloned from E. tarda and expressed in Escherichia coli M15 cells. The recombinant OmpA protein containing His₆ residues was estimated to have a molecular weight of ∼38 kDa. In Western blot the native protein showed expression at ∼36 kDa molecular weight which was within the range of major outer membrane proteins (36–44 kDa) observed in this study. All E. tarda isolates tested harbored the ompA gene and the antibody raised to this protein was seen to cross react with other Gram negative bacteria. The OmpA protein characterized in this study was observed to be highly immunogenic in both rabbit and fish. In Enzyme linked immunosorbent assay, rabbit antisera showed an antibody titer of 1: 128,000. Common carp vaccinated with recombinant OmpA protein elicited high antibody production and immunized fish showed a relative percentage survival of 54.3 on challenge.     

An outer membrane protein A (ompA) homologue from the photosynthetic purple sulfur bacterium Allochromatium vinosum

A 991 bp DNA fragment, consisting of a 225 amino acid reading frame homologous to outer membrane protein coding ompA gene, was cloned from a purple sulfur bacterium Allochromatium vinosum. The homology analysis revealed up to 51% similarity to other bacterial species. The absence of branching within diazotrophs or other taxonomically related groups shows the structural importance of the protein regardless of the metabolism and evolution of the species.     

Folding of outer membrane protein A in the anionic biosurfactant rhamnolipid

Folding and stability of bacterial outer membrane proteins (OMPs) are typically studied in vitro using model systems such as phospholipid vesicles or surfactant. OMP folding requires surfactant concentrations above the critical micelle concentration (cmc) and usually only occurs in neutral or zwitterionic surfactants, but not in anionic or cationic surfactants. Various Gram-negative bacteria produce the anionic biosurfactant rhamnolipid. Here we show that the OMP OmpA can be folded in rhamnolipid at concentrations above the cmc, though the thermal stability is reduced compared to the non-ionic surfactant dodecyl maltoside. We discuss implications for possible interactions between OMPs and biosurfactants in vivo.     

A peptidoglycan binding domain in the porin-associated protein (PAP) of Rhodospirillum rubrum FR1

Abstract The porin-associated protein of Rhodospirillum rubrum FR1 was found to contain a peptidoglycan binding motif. A partial fragment of 179 amino acids, obtained by cleavage of PAP with trypsin, Asp-N protease, and CNBr, was sequenced. Substantial sequence homology was found of the C-terminal part (residues 126–179) of porin-associated protein with OmpA, the peptidoglycan-associated lipoprotein of several bacteria, protein F of Pseudomonas aeruginosa , and PIII of Neisseria gonorrhoeae , the latter being also a porin-associated protein. The 179 amino acid fragment comprised about 67% of the mass spectrometrically determined total mass of PAP of 27 850 Da.     

Structure, dynamics and topology of membrane polypeptides by oriented 2H solid-state NMR spectroscopy

Knowledge of the structure, dynamics and interactions of polypeptides when associated with phospholipid bilayers is key to understanding the functional mechanisms of channels, antibiotics, signal- or translocation peptides. Solid-state NMR spectroscopy on samples uniaxially aligned relative to the magnetic field direction offers means to determine the alignment of polypeptide bonds and domains relative to the bilayer normal. Using this approach the ¹⁵N chemical shift of amide bonds provides a direct indicator of the approximate helical tilt, whereas the ²H solid-state NMR spectra acquired from peptides labelled with 3,3,3-²H₃-alanines contain valuable complimentary information for a more accurate analysis of tilt and rotation pitch angles. The deuterium NMR line shapes are highly sensitive to small variations in the alignment of the C_α–C_β bond relative to the magnetic field direction and, therefore, also the orientational distribution of helices relative to the membrane normal. When the oriented membrane samples are investigated with their normal perpendicular to the magnetic field direction, the rate of rotational diffusion can be determined in a semi-quantitative manner and thereby the aggregation state of the peptides can be analysed. Here the deuterium NMR approach is first introduced showing results from model amphipathic helices. Thereafter investigations of the viral channel peptides Vpu_1–27 and Influenza A M2_22–46 are shown. Whereas the ¹⁵N chemical shift data confirm the transmembrane helix alignments of these hydrophobic sequences, the deuterium spectra indicate considerable mosaic spread in the helix orientations. At least two peptide populations with differing rotational correlation times are apparent in the deuterium spectra of the viral channels suggesting an equilibrium between monomeric peptides and oligomeric channel configurations under conditions where solid-state NMR structural studies of these peptides have previously been performed. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Structure determination of symmetric homo-oligomers by a complete search of symmetry configuration space, using NMR restraints and van der Waals packing

Structural studies of symmetric homo-oligomers provide mechanistic insights into their roles in essential biological processes, including cell signaling and cellular regulation. This paper presents a novel algorithm for homo-oligomeric structure determination, given the subunit structure, that is both complete, in that it evaluates all possible conformations, and data-driven, in that it evaluates conformations separately for consistency with experimental data and for quality of packing. Completeness ensures that the algorithm does not miss the native conformation, and being data-driven enables it to assess the structural precision possible from data alone. Our algorithm performs a branch-and-bound search in the symmetry configuration space, the space of symmetry axis parameters (positions and orientations) defining all possible C(n) homo-oligomeric complexes for a given subunit structure. It eliminates those symmetry axes inconsistent with intersubunit nuclear Overhauser effect (NOE) distance restraints and then identifies conformations representing any consistent, well-packed structure to within a user-defined similarity level. For the human phospholamban pentamer in dodecylphosphocholine micelles, using the structure of one subunit determined from a subset of the experimental NMR data, our algorithm identifies a diverse set of complex structures consistent with the nine intersubunit NOE restraints. The distribution of determined structures provides an objective characterization of structural uncertainty: backbone RMSD to the previously determined structure ranges from 1.07 to 8.85 A, and variance in backbone atomic coordinates is an average of 12.32 A(2). Incorporating vdW packing reduces structural diversity to a maximum backbone RMSD of 6.24 A and an average backbone variance of 6.80 A(2). By comparing data consistency and packing quality under different assumptions of oligomeric number, our algorithm identifies the pentamer as the most likely oligomeric state of phospholamban, demonstrating that it is possible to determine the oligomeric number directly from NMR data. Additional tests on a number of homo-oligomers, from dimer to heptamer, similarly demonstrate the power of our method to provide unbiased determination and evaluation of homo-oligomeric complex structures.     

Characterization of protein detergent complexes by NMR, light scattering, and analytical ultracentrifugation

Abstract  Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably, this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore more effective than centrifugal ultrafiltration devices.     

Comparative characterization of the glycosylation profiles of an influenza hemagglutinin produced in plant and insect hosts

The main objective of this study was to characterize the N-linked glycosylation profiles of recombinant hemagglutinin (HA) proteins expressed in either insect or plant hosts, and to develop a mass spectrometry based workflow that can be used in quality control to assess batch-to-batch reproducibility for recombinant HA glycosylation. HA is a surface glycoprotein of the influenza virus that plays a key role in viral infectivity and pathogenesis. Characterization of the glycans for plant recombinant HA from the viral strain A/California/04/09 (H1N1) has not yet been reported. In this study, N-linked glycosylation patterns of the recombinant HAs from both insect and plant hosts were characterized by precursor ion scan-driven data-dependent analysis followed by high-resolution MS/MS analysis of the deglycosylated tryptic peptides. Five glycosylation sites (N11, N23, N276, N287, and N481) were identified containing high mannose type glycans in plant-expressed HAs, and complex type glycoforms for the insect-expressed HA. More than 95% site occupancy was observed for all glycosylation sites except N11, which was 60% occupied. Multiple-reaction monitoring based quantitation analysis was developed for each glycopeptide isoform and the quantitative results indicate that the Man(8) GlcNAc(2) is the dominant glycan for all sites in plant-expressed HAs. The relative abundance of the glycoforms at each specific glycosylation site and the relative quantitation for each glycoform among three HAs were determined. Few differences in the glycosylation profiles were detected between the two batches of plant HAs studied, but there were significant differences between the glycosylation patterns in the HAs generated in plant and insect expression hosts.     

Solid-state NMR characterization of conformational plasticity within the transmembrane domain of the influenza A M2 proton channel

Membrane protein function within the membrane interstices is achieved by mechanisms that are not typically available to water-soluble proteins. The whole balance of molecular interactions that stabilize three-dimensional structure in the membrane environment is different from that in an aqueous environment. As a result interhelical interactions are often dominated by non-specific van der Waals interactions permitting dynamics and conformational heterogeneity in these interfaces. Here, solid-state NMR data of the transmembrane domain of the M2 protein from influenza A virus are used to exemplify such conformational plasticity in a tetrameric helical bundle. Such data lead to very high resolution structural restraints that can identify both subtle and substantial structural differences associated with various states of the protein. Spectra from samples using two different preparation protocols, samples prepared in the presence and absence of amantadine, and spectra as a function of pH are used to illustrate conformational plasticity.     

NMR dynamics and antibody recognition of the meningococcal lipidated outer membrane protein LP2086 in micellar solution

Neisseria meningitidis is a major cause of meningitis. Although protective vaccination is available against some pathogenic serogroups, serogroup B meningococci have been a challenge for vaccinologists. A family of outer membrane lipoproteins, LP2086 (or factor H binding proteins, fHbp), has been shown to elicit bactericidal antibodies and is currently part of a cocktail vaccine candidate. The NMR structure of the variant LP2086-B01 in micellar solution provided insights on the topology of this family of proteins on the biological membrane. Based on flow cytometry experiments on whole meningococcal cells, binding experiments with monoclonal antibodies, and the NMR structure in micellar solution, we previously proposed that LP2086-B01 anchors the outer bacterial membrane through its lipidated N-terminal cysteine, while a flexible 20 residue linker positions the protein above the layer of lipo-oligosaccharides that surrounds the bacteria. This topology was suggested to increase the antigen exposure to the immune system. In the present work, using micellar solution as a membrane mimicking system, we characterized the backbone dynamics of the variant LP2086-B01 in both its lipidated and unlipidated forms. In addition, binding experiments with a Fab fragment derived from the monoclonal MN86-1042-2 were also performed. Our data suggests that due to the length and flexibility of the N-terminal linker, the antigen is not in contact with the micelle, thus making both N- and C-domains highly available to the host immune system. This dynamic model, combined with the binding data obtained with MN86-1042-2, supports our previously proposed arrangement that LP2086-B01 exposes one face to the extracellular space. Binding of MN86-1042-2 antibody shows that the N-domain is the primary target of this monoclonal, providing further indication that this domain is immunologically important for this family of proteins.     

Expression, purification, and initial structural characterization of nonstructural protein 2B, an integral membrane protein of Dengue-2 virus, in detergent micelles

Dengue virus causes serious diseases affecting people in tropical and sub-tropical regions. The nonstructural (NS) protein 2B is an integral membrane protein and important for the regulation of viral protease NS3, which is significant for virus replication. The NS2B-NS3 complex is an important drug target for treating dengue fever. However, little is known about the structure of NS2B in its entirety. Herein, we describe the expression and purification of this integral membrane protein from cell membrane and inclusion bodies of Escherichia coli cells. The initial nuclear magnetic resonance (NMR) and circular dichroism (CD) results indicate that the purified protein adopts alpha-helical structures in LMPG and TDPC micelles.     

Structure and dynamics of the von Willebrand Factor C6 domain

Von Willebrand disease (VWD) is a bleeding disorder with different levels of severity. VWD-associated mutations are located in the von Willebrand factor (VWF) gene, coding for the large multidomain plasma protein VWF with essential roles in hemostasis and thrombosis. On the one hand, a variety of mutations in the C-domains of VWF are associated with increased bleeding upon vascular injury. On the other hand, VWF gain-of-function (GOF) mutations in the C4 domain have recently been identified, which induce an increased risk of myocardial infarction. Mechanistic insights into how these mutations affect the molecular behavior of VWF are scarce and holistic approaches are challenging due to the multidomain and multimeric character of this large protein. Here, we determine the structure and dynamics of the C6 domain and the single nucleotide polymorphism (SNP) variant G2705R in C6 by combining nuclear magnetic resonance spectroscopy, molecular dynamics simulations and aggregometry. Our findings indicate that this mutation mostly destabilizes VWF by leading to a more pronounced hinging between both subdomains of C6. Hemostatic parameters of variant G2705R are close to normal under static conditions, but the missense mutation results in a gain-of-function under flow conditions, due to decreased VWF stem stability. Together with the fact that two C4 variants also exhibit GOF characteristics, our data underline the importance of the VWF stem region in VWF’s hemostatic activity and the risk of mutation-associated prothrombotic properties in VWF C-domain variants due to altered stem dynamics.