Residual dipolar coupling measurements of transmembrane proteins using aligned low-q bicelles and high-resolution magic angle spinning NMR spectroscopy

Bicelles are a major medium form to produce weak alignment of soluble proteins for residual dipolar coupling (RDC) measurements. The obstacle to using the same type of bicelles for transmembrane proteins with solution-state NMR spectroscopy is the loss of signals due to the adhesion or penetration of the proteins into large bicelles, resulting in slow protein tumbling. In this study, weak alignment of the second and third transmembrane domains (TM23) of the human glycine receptor (GlyR) was achieved in low-q bicelles (q = DMPC/DHPC). Although protein-free bicelles with such low q would likely show isotropic properties, the insertion of TM23 induced weakly preferred orientations so that the RDC of the embedded protein can be measured. The extent of the alignment increased but the TM23 signal intensity decreased when q was varied from 0.19 to 0.60. A q of 0.50 was found to be an optimal compromise between alignment and the signal-to-noise ratio. In each pair of NMR experiments for RDC measurements, the same sample and pulse sequence were used, with one being performed at high-resolution magic-angle spinning to obtain pure J-couplings without RDC. A meaningful structure refinement in bicelles was possible by iteratively fitting the experimental RDCs to the back-calculated RDCs using the high-resolution NMR structure of GlyR TM23 in trifluoroethanol as the starting template. Combination of this method with the conventional high-resolution NMR in membrane mimicking mixtures of water and organic solvents offers an attractive way to derive structural information for membrane proteins in their native environment.     

Increasing the accuracy of solution NMR structures of membrane proteins by application of residual dipolar couplings. High-resolution structure of outer membrane protein A

The structure determination of membrane proteins is one of the most challenging applications of solution NMR spectroscopy. The paucity of distance information available from the highly deuterated proteins employed requires new approaches in structure determination. Here we demonstrate that significant improvement in the structure accuracy of the membrane protein OmpA can be achieved by refinement with residual dipolar couplings (RDCs). The application of charged polyacrylamide gels allowed us to obtain two alignments and accurately measure numerous heteronuclear dipolar couplings. Furthermore, we have demonstrated that using a large set of RDCs in the refinement can yield a structure with 1 A rms deviation to the backbone of the high-resolution crystal structure. Our simulations with various data sets indicate that dipolar couplings will be critical for obtaining accurate structures of membrane proteins.     

Dependence of the size of a protein-SDS complex on detergent and Na+ concentrations

Sodium dodecyl sulfate (SDS) micelles provide ideal mimetic media for high-resolution NMR studies of membrane proteins and proteins or peptides interacting with micellar aggregates. (15)N NMR relaxation of the backbone amides of a protein-SDS complex has been measured under different experimental conditions. The rotational diffusion time of this complex has been found highly sensitive to detergent and NaCl concentrations. A comparison with calculated rotational diffusion times of protein-free SDS micelles under the same conditions suggests that the size of both aggregates must follow a similar functional dependence on detergent/NaCl concentration.     

Membrane orientation of the Na,K-ATPase regulatory membrane protein CHIF determined by solid-state NMR

Corticosteroid hormone-induced factor (CHIF) is a major regulatory subunit of the Na,K-ATPase, and a member of an evolutionarily conserved family of membrane proteins that regulate the function of the enzyme complex in a tissue-specific and physiological-state-specific manner. Here we present the structure of CHIF oriented in the membrane, determined by solid-state NMR orientation-dependent restraints. Because CHIF adopts a similar structure in lipid micelles and bilayers, it is possible to assign the solid-state NMR spectrum measured for (15)N-labeled CHIF in oriented bilayers from the structure determined in micelles, to obtain the global orientation of the protein in the membrane.     

NMR of membrane-associated peptides and proteins

In living cells, membrane proteins are essential to signal transduction, nutrient use, and energy exchange between the cell and environment. Due to challenges in protein expression, purification and crystallization, deposition of membrane protein structures in the Protein Data Bank lags far behind existing structures for soluble proteins. This review describes recent advances in solution NMR allowing the study of a select set of peripheral and integral membrane proteins. Surface-binding proteins discussed include amphitropic proteins, antimicrobial and anticancer peptides, the HIV-1 gp41 peptides, human alpha-synuclein and apolipoproteins. Also discussed are transmembrane proteins including bacterial outer membrane beta-barrel proteins and oligomeric alpha-helical proteins. These structural studies are possible due to solubilization of the proteins in membrane-mimetic constructs such as detergent micelles and bicelles. In addition to protein dynamics, protein-lipid interactions such as those between arginines and phosphatidylglycerols have been detected directly by NMR. These examples illustrate the unique role solution NMR spectroscopy plays in structural biology of membrane proteins.     

Conformational fluctuations affect protein alignment in dilute liquid crystal media

The discovery of dilute liquid crystalline media to align biological macromolecules has opened many new possibilities to study protein and nucleic acid structures by NMR spectroscopy. We inspect the basic alignment phenomenon for an ensemble of protein conformations to deduce relative contributions of each member to the residual dipolar coupling signals. We find that molecular fluctuations can affect the alignment and discover a resulting emphasis of certain conformations. However, the internal fluctuations are largely uncorrelated with those of the alignment, implying that proteins have liquidlike molecular surfaces. Furthermore, we consider the implications of a dynamic bias to structure determination using data from the weak alignment method.     

NMR at 900 MHz

The very first high-resolution NMR spectra recorded at 900 MHz in July 2000 have demonstrated the benefits of increased magnetic field strength for studies of large biomolecules such as proteins and nucleic acids. Increased sensitivity and resolution for such molecules can only be observed in experiments that are optimized for transverse relaxation (TROSY). Substantial effects of magnetic alignment can easily be observed not only in paramagnetic proteins, but even in small molecules, such as chloroform. Such effects can be very useful for structural studies of biopolymers. The extreme resolution allows studies of very weak interactions in proteins. For instance, long-range H/D isotope effects are easily observed in H-N correlation experiments. The first systematic studies of relaxation properties of N-15 nuclei have been carried out for proteins at 500, 600, 800, and 900 MHz.     

Ultrafiltration of sodium dodecylsulfate solutions

The present paper is devoted to ultrafiltration (UF) of aqueous solutions containing micellized sodium dodecylsulfate (NaDS) through zircon membranes, following permeation data of cetyltrimethylammonium bromide (CTABr), which we have reported recently.The experimental results are analyzed using the same model of diffusion in the membrane matrix based on the thermodynamics of irreversible processes (TIP). A comparison with the classical model of resistances in series leads to a new analysis of the measured resistances in terms of membrane–permeant and permeant–permeant interactions. NaDS micelles being able to cross the membrane as permeants, in contrast with CTABr micelles, the comparison of the behaviors of anionic and cationic surfactants is worthwhile. The resistance of the membrane strongly depends on the composition of the feed. The TIP approach allows us to relate the membrane resistance to the concentrations of all the species present in the feed, namely free ions, spherical micelles and rod-like aggregates. It is shown that the resistance is highly influenced by free ions (dodecylsulfate (DS⁻) and sodium (Na⁺)), very slightly by negatively charged spherical micelles, and weakly by rod-like aggregates, also negatively charged.     

Optical spectroscopic and TEM studies of catanionic micelles of CTAB/SDS and their interaction with a NSAID

If a vesicle is a better model of a membrane in the context of the hydrophobic effect, then from the charge distribution point of view, a catanionic micelle is a closer model to a biomembrane. We have prepared and characterized two different types of catanionic micelles of sodium dodecyl sulfate (SDS) and cetyl N,N,N-trimethylammonium bromide (CTAB) having different surface charge ratios using optical spectroscopy and transmission electron microscopy. The average size of both types of mixed micelles was found to be much larger than that of micelles containing uniformly charged headgroups. Catanionic micelles containing higher concentrations of positively charged headgroups (CTAB) are larger in size, less compact, and more polar compared to the micelles containing higher concentrations of negatively charged headgroups (SDS). We have used these catanionic micelles as membrane mimetic systems to understand the interaction of piroxicam, a nonsteroidal anti-inflammatory drug (NSAID) of the oxicam group, with biomembranes. In continuation of our work on membrane mimetic systems, we have used spectral properties of the drug itself to understand the effect of the presence of mixed charges on the micellar surface in guiding the interaction of catanionic micelles with piroxicam. Our earlier studies of the interaction of piroxicam with micelles having uniform surface charges have shown that the charge on the micellar surface not only dictates which prototropic form of the drug will be incorporated in the micelles but also induces a switch-over between different prototropic forms of piroxicam. The equilibrium of this switch-over is extremely sensitive to the environment. In this study, we demonstrate how even small changes in the electrostatic forces obtained by doping the uniformly charged surface of the micelles with oppositely charged headgroups (as in catanionic micelles) are capable of fine-tuning this equilibrium. This implies that the surface charge of biomembranes, which are quite diverse in vivo, might play a significant role in selecting a particular form of the drug to be presented to its targets.     

Paramagnetic-based NMR restraints lift residual dipolar coupling degeneracy in multidomain detergent-solubilized membrane proteins

Residual dipolar couplings (RDCs) are widely used as orientation-dependent NMR restraints to improve the resolution of the NMR conformational ensemble of biomacromolecules and define the relative orientation of multidomain proteins and protein complexes. However, the interpretation of RDCs is complicated by the intrinsic degeneracy of analytical solutions and protein dynamics that lead to ill-defined orientations of the structural domains (ghost orientations). Here, we illustrate how restraints from paramagnetic relaxation enhancement (PRE) experiments lift the orientational ambiguity of multidomain membrane proteins solubilized in detergent micelles. We tested this approach on monomeric phospholamban (PLN), a 52-residue membrane protein, which is composed of two helical domains connected by a flexible loop. We show that the combination of classical solution NMR restraints (NOEs and dihedral angles) with RDC and PRE constraints resolves topological ambiguities, improving the convergence of the PLN structural ensemble and giving the depth of insertion of the protein within the micelle. The combination of RDCs with PREs will be necessary for improving the accuracy and precision of membrane protein conformational ensembles, where three-dimensional structures are dictated by interactions with the membrane-mimicking environment rather than compact tertiary folds common in globular proteins.     

Bioinspired,Size‐Tunable Self‐Assembly of Polymer–Lipid Bilayer Nanodiscs

Polymer‐based nanodiscs are valuable tools in biomedical research that can offer a detergent‐free solubilization of membrane proteins maintaining their native lipid environment. Herein, we introduce a novel ca. 1.6 kDa SMA‐based polymer with styrene:maleic acid moieties that can form nanodiscs containing a planar lipid bilayer which are useful to reconstitute membrane proteins for structural and functional studies. The physicochemical properties and the mechanism of formation of polymer‐based nanodiscs are characterized by light scattering, NMR, FT‐IR, and TEM. A remarkable feature is that nanodiscs of different sizes, from nanometer to sub‐micrometer diameter, can be produced by varying the lipid‐to‐polymer ratio. The small‐size nanodiscs (up to ca. 30 nm diameter) can be used for solution NMR spectroscopy studies whereas the magnetic‐alignment of macro‐nanodiscs (diameter of > ca. 40 nm) can be exploited for solid‐state NMR studies on membrane proteins.     

Bioinspired,Size‐Tunable Self‐Assembly of Polymer–Lipid Bilayer Nanodiscs

Polymer‐based nanodiscs are valuable tools in biomedical research that can offer a detergent‐free solubilization of membrane proteins maintaining their native lipid environment. Herein, we introduce a novel ca. 1.6 kDa SMA‐based polymer with styrene:maleic acid moieties that can form nanodiscs containing a planar lipid bilayer which are useful to reconstitute membrane proteins for structural and functional studies. The physicochemical properties and the mechanism of formation of polymer‐based nanodiscs are characterized by light scattering, NMR, FT‐IR, and TEM. A remarkable feature is that nanodiscs of different sizes, from nanometer to sub‐micrometer diameter, can be produced by varying the lipid‐to‐polymer ratio. The small‐size nanodiscs (up to ca. 30 nm diameter) can be used for solution NMR spectroscopy studies whereas the magnetic‐alignment of macro‐nanodiscs (diameter of > ca. 40 nm) can be exploited for solid‐state NMR studies on membrane proteins.     

Solution NMR spectroscopy of the human vasopressin V2 receptor, a G protein-coupled receptor

The seven-transmembrane-spanning G protein-coupled receptor (GPCR) superfamily plays many important roles in basic biology, human health, and human disease. Here, well-resolved solution NMR spectra are presented for a human GPCR, the vasopressin V2 receptor in detergent micelles. The quality of the NMR spectra indicates that backbone resonance assignments for a majority of resonances are feasible. The key to obtaining high quality spectra appears to be the coupling of methods for expressing the receptor into membranes rather than into inclusion bodies, with use of a biochemically mild lysolipid detergent for membrane extraction, protein purification, and NMR sample preparation.     

Solid-state NMR spectroscopy of aligned lipid bilayers at low temperatures

This study, for the first time, demonstrates that it is possible to mechanically align lipid bilayers at a very low temperature (as low as the gel-to-liquid crystalline phase transition temperature). Performing NMR experiments on mechanically aligned lipid bilayers at a low temperature increases the signal-to-noise ratio, the resolution, and the span of NMR parameters. The increased lifetime of the alignment and the nature of the bilayer sample would enhance the application of solid-state NMR techniques to study membrane proteins.     

Collapse of polyelectrolyte networks induced by their interaction with an oppositely charged surfactant. Theory

A very simple theory of swelling and collapse of weakly charged polyelectrolyte networks in the solution of an oppositely charged surfactant has been developed. The following contributions to the free energy were taken into account: free energy of volume interaction and of elastic deformation of the network chains, free energy connected with micelle formation and free energy of translational motion of all mobile ions in the system (translational entropy). Both the cases of a solution of charged surfactant and that of a mixed solution of charged and neutral surfactant components have been taken into account. It has been shown that the behaviour of the network depends on the total surfactant concentration in the system and corresponds to one of the three following regimes: At low concentration, micelles inside the network are not formed and the behaviour of the polymer network is similar to that of a network swelling in the solution of a lowmolecular-weight salt (regime 1). In the second regime, surfactant concentration inside the network exceeds the critical micelle concentration and micelles are formed; in this regime the network collapses because surfactant molecules, aggregated in micelles, cease to create “exerting” osmotic pressure in the network sample. In the third regime, at very high surfactant concentration, formation of additional micelles inside the network ceases, and the network dimensions coincide with those of the corresponding neutral network.     

Solution NMR approaches for establishing specificity of weak heterodimerization of membrane proteins

Solution NMR provides a powerful approach for detecting complex formation involving weak to moderate intermolecular affinity. However, solution NMR has only rarely been used to detect complex formation between two membrane proteins in model membranes. The impact of specific binding on the NMR spectrum of a membrane protein can be difficult to distinguish from spectral changes that are induced by nonspecific binding and/or by changes that arise from forced cohabitation of the two proteins in a single model membrane assembly. This is particularly the case when solubility limits make it impossible to complete a titration to the point of near saturation of complex formation. In this work experiments are presented that provide the basis for establishing whether specific complex formation occurs between two membrane proteins under conditions where binding is not of high avidity. Application of these methods led to the conclusion that the membrane protein CD147 (also known as EMMPRIN or basigin) forms a specific heterodimeric complex in the membrane with the 99-residue transmembrane C-terminal fragment of the amyloid precursor protein (C99 or APP-βCTF), the latter being the immediate precursor of the amyloid-β polypeptides that are closely linked to the etiology of Alzheimer's disease.     

Elongated Bacterial Pili as a Versatile Alignment Medium for NMR Spectroscopy

In NMR spectroscopy, residual dipolar couplings (RDCs) have emerged as one of the most exquisite probes of biological structure and dynamics. The measurement of RDCs relies on the partial alignment of the molecule of interest, for example by using a liquid crystal as a solvent. Here, we establish bacterial type 1 pili as an alternative liquid-crystalline alignment medium for the measurement of RDCs. To achieve alignment at pilus concentrations that allow for efficient NMR sample preparation, we elongated wild-type pili by recombinant overproduction of the main structural pilus subunit. Building on the extraordinary stability of type 1 pili against spontaneous dissociation and unfolding, we show that the medium is compatible with challenging experimental conditions such as high temperature, the presence of detergents, organic solvents or very acidic pH, setting it apart from most established alignment media. Using human ubiquitin, HIV-1 TAR RNA and camphor as spectroscopic probes, we demonstrate the applicability of the medium for the determination of RDCs of proteins, nucleic acids and small molecules. Our results show that type 1 pili represent a very useful alternative to existing alignment media and may readily assist the characterization of molecular structure and dynamics by NMR.     

Self-assembly of flexible β-strands into immobile amyloid-like β-sheets in membranes as revealed by solid-state 19F NMR

The cationic peptide [KIGAKI](3) was designed as an amphiphilic β-strand and serves as a model for β-sheet aggregation in membranes. Here, we have characterized its molecular conformation, membrane alignment, and dynamic behavior using solid-state (19)F NMR. A detailed structure analysis of selectively (19)F-labeled peptides was carried out in oriented DMPC bilayers. It showed a concentration-dependent transition from monomeric β-strands to oligomeric β-sheets. In both states, the rigid (19)F-labeled side chains project straight into the lipid bilayer but they experience very different mobilities. At low peptide-to-lipid ratios ≤1:400, monomeric [KIGAKI](3) swims around freely on the membrane surface and undergoes considerable motional averaging, with essentially uncoupled φ/ψ torsion angles. The flexibility of the peptide backbone in this 2D plane is reminiscent of intrinsically unstructured proteins in 3D. At high concentrations, [KIGAKI](3) self-assembles into immobilized β-sheets, which are untwisted and lie flat on the membrane surface as amyloid-like fibrils. This is the first time the transition of monomeric β-strands into oligomeric β-sheets has been characterized by solid-state NMR in lipid bilayers. It promises to be a valuable approach for studying membrane-induced amyloid formation of many other, clinically relevant peptide systems.     

Sequence-specific resonance assignment of soluble nonglobular proteins by 7D APSY-NMR spectroscopy

Based on sequence-specific resonance assignments, NMR is the method of choice for obtaining atomic-resolution experimental data on soluble nonglobular proteins. So far, however, NMR assignment of unfolded polypeptides in solution has been a time-consuming task, mainly due to the small chemical shift dispersion, which has limited practical applications of the NMR approach. This paper presents an efficient, fully automated method for sequence-specific backbone and beta-carbon NMR assignment of soluble nonglobular proteins with sizes up to at least 150 residues. The procedure is based on new APSY (automated projection spectroscopy) experiments which benefit from the short effective rotational correlation times in soluble nonglobular polypeptides to record five- to seven-dimensional NMR data sets, which reliably resolves chemical shift degeneracies. Fully automated sequence-specific resonance assignments of the backbone nuclei and C(beta) are described for the uniformly (13)C,(15)N-labeled urea-denatured 148-residue outer membrane protein X (OmpX) from E. coli. The method is generally applicable to systems with similar spectroscopic properties as unfolded OmpX, and we anticipate that this paper may open the door for extensive atomic-resolution studies of chemical denaturant-unfolded proteins, as well as some classes of functional nonglobular polypeptides in solution.     

Three-dimensional structure of the water-insoluble protein crambin in dodecylphosphocholine micelles and its minimal solvent-exposed surface

We chose crambin, a hydrophobic and water-insoluble protein originally isolated from the seeds of the plant Crambe abyssinica, as a model for NMR investigations of membrane-associated proteins. We produced isotopically labeled crambin(P22,L25) (variant of crambin containing Pro22 and Leu25) as a cleavable fusion with staphylococcal nuclease and refolded the protein by an approach that has proved successful for the production of proteins with multiple disulfide bonds. We used NMR spectroscopy to determine the three-dimensional structure of the protein in two membrane-mimetic environments: in a mixed aqueous-organic solvent (75%/25%, acetone/water) and in DPC micelles. With the sample in the mixed solvent, it was possible to determine (>NH...OC<) hydrogen bonds directly by the detection of (h3)J(NC)' couplings. H-bonds determined in this manner were utilized in the refinement of the NMR-derived protein structures. With the protein in DPC (dodecylphosphocholine) micelles, we used manganous ion as an aqueous paramagnetic probe to determine the surface of crambin that is shielded by the detergent. With the exception of the aqueous solvent exposed loop containing residues 20 and 21, the protein surface was protected by DPC. This suggests that the protein may be similarly embedded in physiological membranes. The strategy described here for the expression and structure determination of crambin should be applicable to structural and functional studies of membrane active toxins and small membrane proteins.