Solution Nuclear Magnetic Resonance Studies Research Articles

Exploring Host-Guest Interactions within a 600 kDa DegP Protease Cage Complex Using Hydrodynamics Measurements and Methyl-TROSY NMR.

The DegP protease-chaperone operates within the periplasm of Gram-negative bacteria, where it assists in the regulation of protein homeostasis, promotes virulence, and is essential to survival under stress. To carry out these tasks, DegP forms a network of preorganized apo oligomers that facilitate the capture of substrates within distributions of cage-like complexes which expand to encapsulate clients of various sizes. Although the architectures of DegP cage complexes are well understood, little is known about the structures, dynamics, and interactions of client proteins within DegP cages and the relationship between client structural dynamics and function. Here, we probe host-guest interactions within a 600 kDa DegP cage complex throughout the DegP activation cycle using a model α-helical client protein through a combination of hydrodynamics measurements, methyl-transverse relaxation optimized spectroscopy-based solution nuclear magnetic resonance studies, and proteolytic activity assays. We find that in the presence of the client, DegP cages assemble cooperatively with few intermediates. Our data further show that the N-terminal half of the bound client, which projects into the interior of the cages, is predominantly unfolded and flexible, and exchanges between multiple conformational states over a wide range of time scales. Finally, we show that a concerted structural transition of the protease domains of DegP occurs upon client engagement, leading to activation. Together, our findings support a model of DegP as a highly cooperative and dynamic molecular machine that stabilizes unfolded states of clients, primarily via interactions with their C-termini, giving rise to efficient cleavage.

Solution NMR studies reveal novel CLIC4 chaperone activity with respect to profilin-1.

Despite its name, chloride intracellular channel 4 (CLIC4) is a soluble enzyme that can catalyze glutathione-dependent thiol transfers. It is unclear how this enzymatic activity relates to its biological role in remodeling the actin cytoskeleton. Previous studies have shown that CLIC4 interacts with profilin-1, a critical regulator of actin polymerization. We confirmed a direct interaction, 170 ± 20 μM by microscale thermophoresis. However, using solution nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that CLIC4 has a higher affinity for profilin when it is unfolded. Amide hydrogen exchange studies demonstrate that the N- and C-terminal helices of profilin readily unfold, whereas the central β-beta sheet core remains relatively intact. Interaction with CLIC4 increases amide exchange rates throughout profilin, indicating preferential stabilization of partially unfolded intermediates. We note that profilin is glutathionylated in proteomic studies of cells exposed to oxidative stress. Profilin is unfolded by glutathionylation of buried cysteine residues in its N- or C-terminal helices, and this disrupts its ability to bind actin, as measured by pyrene-actin polymerization assay. We are currently attempting to demonstrate that CLIC4 can reverse glutathionylation of profilin-1 and promote its refolding in cells and in vitro.

One-bond 13C-13C spin-coupling constants in saccharides: a comparison of experimental and calculated values by density functional theory using solid-state 13C NMR and X-ray crystallography.

Methyl aldohexopyranosides were 13C-labeled at contiguous carbons, crystallized, and studied by single-crystal X-ray crystallography and solid-state 13C nuclear magnetic resonance (NMR) spectroscopy to examine the degree to which density functional theory (DFT) can calculate one-bond 13C-13C spin-coupling constants (1JCC) in saccharides with sufficient accuracy to permit their use in MA'AT analysis, a newly-reported hybrid DFT/NMR method that provides probability distributions of molecular torsion angles in solution (Zhang et al., J. Phys. Chem. B, 2017, 121, 3042-3058; Meredith et al., J. Chem. Inf. Model., 2022, 62, 3135-3141). Experimental 1JCC values in crystalline samples of the doubly 13C-labeled compounds were measured by solid-state 13C NMR and compared to those calculated from five different DFT models: (1) 1JCC values calculated from single structures identical to those observed in crystalline samples by X-ray crystallography (all atom refinement); (2) 1JCC values calculated from the single structures in (1) but after Hirshfeld atom refinement (HAR); (3) 1JCC values calculated from the single structures in (1) after DFT-optimization of hydrogen atoms only; and (4 and 5) 1JCC values calculated in rotamers of torsion angle θ2 (C1-C2-O2-O2H) or ω (C4-C5-C6-O6) from which either specific or generalized parameterized equations were obtained and used to calculate 1JCC values in the specific θ2 or ω rotamers observed in crystalline samples. Good qualitative agreement was observed between calculated 1JCC values and those measured by solid-state 13C NMR regardless of the DFT model, but in no cases were calculated 1JCC values quantitative, differing (over-estimated) on average by 4-5% from experimental values. These findings, and those reported recently from solution NMR studies (Tetrault et al., J. Phys. Chem. B 2022, 126, 9506-9515), indicate that improvements in DFT calculations are needed before calculated 1JCC values can be used directly as reliable constraints in MA'AT analyses of saccharides in solution.

Utility of methyl side chain probes for solution NMR studies of large proteins

Selective isotopic labeling of methyl side chain groups in proteins and other biomolecules, combined with advances in perdeuteration, new pulse sequences, and high field spectrometers with cryogenic probes, has revolutionized the field of solution nuclear magnetic resonance (NMR) spectroscopy by enabling characterization of macromolecular systems with molecular weights above 1 MDa in their native aqueous environment. This tutorial provides a basic overview for how modern NMR spectroscopists can utilize methyl side chain probes to study their system of interest. The advantages and limitations of methyl side chain probes are discussed. In addition, the preparation of selectively 13C-methyl labeled recombinant protein samples, strategies for manual and automated methyl NMR resonance assignment, and the application of methyl probes for characterization of dynamics and conformational exchange are discussed. A sneak preview for ways in which methyl probes are expected to continue to advance the field of biomolecular NMR towards new horizons in solution studies of large supramolecular complexes is also presented.

Competitor hydrogen-bond acceptors in the SP(NH)3-based structures: Comparison of structural features – Computational/database and experimental

The phosphorothioic triamides (4-Cl-C6H4CH2NH)3P(S) (I) and (4-CH3-C6H4CH2NH)3P(S) (II) were synthesized to study their coordination behaviors towards Hg(II) cation. The resulting complexes, [(4-Cl-C6H4CH2NH)3P(S)]2Hg2Cl4 (III) and [(4-CH3-C6H4CH2NH)3P(S)]2Hg2Cl4 (IV), are the first examples of mercury complexes with SP(NHR)3 ligands characterized by X-ray crystallography. The presence of NH/CH units and some potential competitor acceptors with moderate/weak acceptor capabilities (sulfur, chlorine and π system) provides the opportunity to study the hydrogen bond elements of (I), (III) and (IV), structurally and of all four compounds theoretically. The strengths of inter- and intramolecular hydrogen bonds were assessed by quantum chemical calculations (Atoms In Molecules (AIM), and Natural Bond Orbital (NBO)), and the internal interactions were further examined by reduced density gradient (RDG) analysis. The differences/similarities of structures were addressed in the view point of geometry, conformations related to flexible substituted benzyl moieties, crowding in the structures and non-bonded contacts, stabilization of four-membered Hg2Cl2 rings and some topics related to solution nuclear magnetic resonance studies (chemical shifts and coupling constants). The similarity of structures investigated allowed for a precise assignment of vibrational frequencies. A survey of the Hg—SP segment in the Cambridge Structural Database shows that the Hg—S bond lengths in (III) and (IV) are among the smallest Hg—S bonds in the structures with the thiophosphoryl-holding ligands.

Amplification of the Specific Conformational Fluctuation of Proteins by Site-Specific Mutagenesis and Hydrostatic Pressure.

Conformational fluctuation, namely, protein interconversion between different conformations, is crucial to protein function. Outer surface protein A (OspA), comprising N- and C-terminal globular domains linked by a central β-sheet, is expressed on the surface of Borrelia burgdorferi, the causative agent of Lyme disease, and recognizes the TROSPA receptor in the tick gut. Solution nuclear magnetic resonance studies have shown that the central β-sheet and C-terminal domain containing TROSPA recognition sites are less stable than the N-terminal domain, revealing an intermediate conformation between the basic folded and completely unfolded proteins. We previously suggested that exposure of receptor-binding sites following denaturation of the C-terminal domain is advantageous for OspA binding to the receptor. Here, we observed amplification of a specific protein fluctuation by pressure perturbation and site-specific mutagenesis. The salt-bridge-destabilized mutant E160D and the cavity-enlarged mutant I243A favored the intermediate. The proportion of the intermediate accounted for almost 100% in E160D at 250 MPa. Strategies using a suitably chosen point mutation with high pressure are generally applicable for amplification of specific conformational fluctuation and potentially improve our understanding of the intermediate conformations of proteins. Knowledge of various conformations, including OspA intermediates, may be useful for designing a vaccine for Lyme disease.

Bacterial Cell Wall Analogue Peptides Control the Oligomeric States and Activity of the Glycopeptide Antibiotic Eremomycin: Solution NMR and Antimicrobial Studies.

For some time, glycopeptide antibiotics have been considered the last line of defense against Methicillin-resistant Staphylococcus aureus (MRSA). However, vancomycin resistance of Gram-positive bacteria is an increasingly emerging worldwide health problem. The mode of action of glycopeptide antibiotics is essentially the binding of peptidoglycan cell-wall fragments terminating in the d-Ala-d-Ala sequence to the carboxylate anion binding pocket of the antibiotic. Dimerization of these antibiotics in aqueous solution was shown to persist and even to enhance the antibacterial effect in a co-operative manner. Some works based on solid state (ss) Nuclear Magnetic Resonance (NMR) studies questioned the presence of dimers under the conditions of ssNMR while in a few cases, higher-order oligomers associated with contiguous back-to-back and face-to-face dimers were observed in the crystal phase. However, it is not proved if such oligomers persist in aqueous solutions. With the aid of 15N-labelled eremomycin using 15N relaxation and diffusion NMR methods, we observed tetramers and octamers when the N-Ac-d-Ala-d-Ala dipeptide was added. To the contrary, the N-Ac-d-Ala or (N-Ac)2-l-Lys-d-Ala-d-Ala tripeptide did not induce higher-order oligomers. These observations are interesting examples of tailored supramolecular self-organization. New antimicrobial tests have also been carried out with these self-assemblies against MRSA and VRE (resistant) strains.

Structural Fingerprints of an Intact Monoclonal Antibody Acquired under Formulated Storage Conditions via 15N Direct Detection Nuclear Magnetic Resonance.

Noninvasive evaluation of tertiary structures is fundamental to the research, development, and use of the biologics. However, few methodologies are currently available for evaluating large molecular weight (MW) biologics, such as therapeutic monoclonal antibodies (mAbs; 150 kDa). Here, we have newly developed a 15N direct detection nuclear magnetic resonance (NMR) technique, the 15N direct detection CRINEPT, which allows the observation of the main chain amide resonances of a nondeuterated protein with MW 150 kDa. The technique not only substantially expands the range of proteins applicable to solution NMR studies but also allows the noninvasive structural analyses of intact mAbs in a wide range of temperature and solvent conditions. As a proof of principle, we successfully acquired the 15N-detected CRINEPT spectra of an intact mAb in its formulated solution at 4 °C. The technique was able to discriminate heterogeneous galactosylation states, demonstrating the benefit of high resolution of the 15N direct detection.

Insights into Structures and Dynamics of Flavivirus Proteases from NMR Studies.

Nuclear magnetic resonance (NMR) spectroscopy plays important roles in structural biology and drug discovery, as it is a powerful tool to understand protein structures, dynamics, and ligand binding under physiological conditions. The protease of flaviviruses is an attractive target for developing antivirals because it is essential for the maturation of viral proteins. High-resolution structures of the proteases in the absence and presence of ligands/inhibitors were determined using X-ray crystallography, providing structural information for rational drug design. Structural studies suggest that proteases from Dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV) exist in open and closed conformations. Solution NMR studies showed that the closed conformation is predominant in solution and should be utilized in structure-based drug design. Here, we reviewed solution NMR studies of the proteases from these viruses. The accumulated studies demonstrated that NMR spectroscopy provides additional information to understand conformational changes of these proteases in the absence and presence of substrates/inhibitors. In addition, NMR spectroscopy can be used for identifying fragment hits that can be further developed into potent protease inhibitors.

Membrane mimetics for solution NMR studies of membrane proteins

AbstractMembrane proteins are one of the most challenging and attractive objects in modern structural biology, as they are targets for the majority of medicines. However, studies of membrane proteins are hindered by several obstacles, including their low ability to crystallize, highly dynamic behavior of some of their domains, and need for membrane-like environment. Although solution nuclear magnetic resonance (NMR) is a very powerful technique of structural biology in terms of the amount of provided data, it imposes several limitations on the object under investigation, with the main constraint being related to the size of the object. For this reason, the membrane mimetic has to form particles of small size and simultaneously to properly simulate the bilayer membrane to be applicable for solution NMR spectroscopy. Here we review the recent advances in the field of membrane mimetics for solution NMR studies, discuss the advantages and drawbacks of specific membrane-like environments, and formulate the criteria for the selection of proper environment for a particular membrane protein or domain.

From Nanodiscs to Isotropic Bicelles: A Procedure for Solution Nuclear Magnetic Resonance Studies of Detergent-Sensitive Integral Membrane Proteins

Nanodiscs and isotropic bicelles are promising membrane mimetics in the field of solution nuclear magnetic resonance (NMR) spectroscopy of integral membrane proteins (IMPs). Despite varied challenges to solution NMR studies of IMPs, we attribute the paucity of solution NMR structures in these environments to the inability of diverse IMPs to withstand detergent treatment during standard nanodisc and bicelle preparations. Here, we present a strategy that creates small isotropic bicelles from IMPs co-translationally embedded in large nanodiscs using cell-free expression. Our results demonstrate appreciable gains in NMR spectral quality while preserving lipid-IMP contacts. We validate the approach on the detergent-sensitive LspA, which finally allowed us to perform high-quality triple-resonance NMR experiments for structural studies. Our strategy of producing bicelles from nanodiscs comprehensively avoids detergent during expression and preparation and is suitable for solution NMR spectroscopy of lipid-IMP complexes.

Structural Insight into Fungal Cell Wall Recognition by a CVNH Protein with a Single LysM Domain

MGG_03307 is a lectin isolated from Magnaporte oryzae, a fungus that causes devastating rice blast disease. Its function is associated with protecting M.oryzae from the host immune response in plants. To provide the structural basis of how MGG_03307 protects the fungus, crystal structures of its CVNH-LysM module were determined in the absence and presence of GlcNAc-containing cell wall chitin constituents, which can act as pathogen-associated molecular patterns. Our structures revealed that glycan binding is accompanied by a notable conformational change in the LysM domain and that GlcNAc3 and GlcNAc4 are accommodated similarly. GlcNAc5 and GlcNAc6 interact with the LysM domain in multiple conformations, as evidenced by solution nuclear magnetic resonance studies. No dimerization of MoCVNH3 via its LysM domain was observed upon binding to GlcNAc6, unlike in multiple LysM domain-containing proteins. Importantly, we define a specific consensus binding mode for the recognition of GlcNAc oligomers by single LysM domains.

Structural Assignment of Side Chain Liquid Crystalline Monomer and Polymer by 1-D and 2-D Solution NMR Studies

In the present study, the synthesis and characterization of a methacrylic-based side chain liquid crystalline monomer and its polymer were investigated with the aid of both 1-D and 2-D nuclear magnetic resonance (NMR) techniques. The mesophase characteristics of the monomer and polymer were determined by hot-stage optical polarized microscopy (HOPM). The nematic and smectic phases were observed for the monomer and polymer. Furthermore, 13C spin-lattice relaxation time measurements were performed on the above systems in order to monitor molecular dynamics. The present study provides an opportunity to carry out a systematic comparison of the evolution of structural as well as dynamical changes of the monomer and the polymer.

Optimized Reverse Micelle Surfactant System for High-Resolution NMR Spectroscopy of Encapsulated Proteins and Nucleic Acids Dissolved in Low Viscosity Fluids

An optimized reverse micelle surfactant system has been developed for solution nuclear magnetic resonance studies of encapsulated proteins and nucleic acids dissolved in low viscosity fluids. Comprising the nonionic 1-decanoyl-rac-glycerol and the zwitterionic lauryldimethylamine-N-oxide (10MAG/LDAO), this mixture is shown to efficiently encapsulate a diverse set of proteins and nucleic acids. Chemical shift analyses of these systems show that high structural fidelity is achieved upon encapsulation. The 10MAG/LDAO surfactant system reduces the molecular reorientation time for encapsulated macromolecules larger than ∼20 kDa leading to improved overall NMR performance. The 10MAG/LDAO system can also be used for solution NMR studies of lipid-modified proteins. New and efficient strategies for optimization of encapsulation conditions are described. 10MAG/LDAO performs well in both the low viscosity pentane and ultralow viscosity liquid ethane and therefore will serve as a general surfactant system for initiating solution NMR studies of proteins and nucleic acids.

Probing Dynamic Conformations of the High-Molecular-Weight αB-Crystallin Heat Shock Protein Ensemble by NMR Spectroscopy

Solution- and solid-state nuclear magnetic resonance (NMR) spectroscopy are highly complementary techniques for studying supra-molecular structure. Here they are employed for investigating the molecular chaperone αB-crystallin, a polydisperse ensemble of between 10 and 40 identical subunits with an average molecular mass of approximately 600 kDa. An IxI motif in the C-terminal region of each of the subunits is thought to play a critical role in regulating the size distribution of oligomers and in controlling the kinetics of subunit exchange between them. Previously published solid-state NMR and X-ray results are consistent with a bound IxI conformation, while solution NMR studies provide strong support for a highly dynamic state. Here we demonstrate through FROSTY (freezing rotational diffusion of protein solutions at low temperature and high viscosity) MAS (magic angle spinning) NMR that both populations are present at low temperatures (<0 °C), while at higher temperatures only the mobile state is observed. Solution NMR relaxation dispersion experiments performed under physiologically relevant conditions establish that the motif interchanges between flexible (highly populated) and bound (sparsely populated) states. This work emphasizes the importance of using multiple methods in studies of supra-molecules, especially for highly dynamic ensembles where sample conditions can potentially affect the conformational properties observed.

The magic of bicelles lights up membrane protein structure.

1.1. Why Study Membranes and Membrane Proteins? Biological membranes and membrane proteins, responsible for numerous exciting biological processes, present one of the paramount challenges in biophysics today. Membranes are present in great number and variety in all organisms. They form the boundary between the inside and outside for any bacterium or cell, and they delimit the host of organelles that make up their inner subunits. Each biological membrane is made up of dozens of different types of lipids and sterols, and any particular type of membrane has a characteristic content of these different constituents. As a very basic example, we mention that prokaryotic membranes contain a notable component of negatively charged lipids but almost no cholesterol, while eukaryotic membranes are mostly zwitterionic but have a significant amount of cholesterol. Since the driving biophysical principles of membrane formation are very simple—they lie in the amphipathic properties of any lipid molecule—a single lipid type is sufficient to form membrane-like bilayers in an aqueous environment. Such model membranes are used extensively to study biophysical properties that are representative for most membrane systems. A particularly interesting effect is observed when detergent molecules are added to lipid bilayer samples: the detergents solubilize the bilayers, and in certain regimes so-called bilayered mixed micelles or “bicelles” are formed. In the simplest case, they can be described as microscopic disks where a bilayer patch is encircled by a “rim” of detergent molecules. Bicelles represent a new instance of lipid morphology and are extensively applicable to structural studies of lipid membranes and protein structure.1 Membranes delimit any cell and all of its compartments. They form natural borders for metabolic substances and signaling molecules. Membrane proteins are the porters and gatekeepers that make sure that only proper molecules or signals make it across the membrane. Since membrane proteins perform numerous key functions in cell metabolism and signaling, they contribute over 30% of the genes in typical eukaryotic genomes,2 and they form the targets for over 50% of drugs in use today.3 The number of elucidated structures of membrane proteins has grown exponentially after the first structure was published in 1985, thus equaling the rate at which structure determination of soluble proteins emerged early on.4 Still, the number of available high-resolution structures of membrane proteins is limited. There are Internet sites that keep track of newly published structures of membrane proteins. The crystallography-oriented Web site of Dr. Stephen White [http://blanco.biomol.uci.edu/mpstruc] has recently been joined by another site maintained by Dr. Dror Warschawski that is dedicated to structures of membrane proteins elucidated by nuclear magnetic resonance (NMR) spectroscopy [www.drorlist.com/nmr/MPNMR.html]. Another equally important site of Dr. Hartmut Michel [www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.html] with an emphasis on crystallization conditions is no longer updated, but states that access is still enabled. In this review article, we aim to give a general overview of lipid bicelles as employed in the study of protein structure. Recent advances in the field of protein structural biology that have been made possible by exploiting the unique properties of lipid bicelles, in both solution and solid-state NMR spectroscopy, will be discussed. During the last five years, review contributions have presented bicelles either within the far more general context of reconstitution media for solution NMR studies (see section 1.4) or have focused on macroscopically aligned bicelles as used for solid-state NMR studies.5,6 One very recent contribution has tackled the formidable task of reviewing all membrane mimetics employed in both solution and solid-state NMR studies.7 As mentioned above, we will limit the contents of this review article to applications of lipid bicelles, but will cover both the isotropic and the aligned bicelles as used in NMR studies. Some parts of this article can be viewed as an update on the review articles of Opella and Marassi,8 Marcotte and Auger,9 and Prosser et al.10 In addition, some of our own recent research involving bicelles is presented in detail.

Solution NMR studies of periplasmic binding proteins and their interaction partners.

Periplasmic binding proteins (PBPs) are a crucial part of ATP-binding cassette import systems in Gram-negative bacteria. Central to their function is the ability to undergo a large-scale conformational rearrangement from open-unliganded to closed-liganded, which signals the presence of substrate and starts its translocation. Over the years, PBPs have been extensively studied not only owing to their essential role in nutrient uptake but also because they serve as excellent models for both practical applications (e.g., biosensor technology) and basic research (e.g., allosteric mechanisms). Although much of our knowledge at atomic level has been inferred from the detailed, static pictures afforded by crystallographic studies, nuclear magnetic resonance (NMR) has been able to fill certain gaps in such body of work, particularly with regard to dynamic processes. Here, we review NMR studies on PBPs, and their unique insights on conformation, dynamics, energetics, substrate binding, and interactions with related transport proteins. Based on the analysis of recent paramagnetic NMR results, as well as crystallographic and functional observations, we propose a mechanism that could explain the ability of certain PBPs to achieve a closed conformation in absence of ligand while others seem to remain open until ligand-mediated closure.

Recombinant expression, purification, and characterization of transmembrane domain of syndecan-4

The transmembrane heparan sulfate proteoglycan, syndecan-4, plays potentially important roles that may influence tissue development and repair as well as the pathogenesis of numerous diseases. Recently, a number of studies have been conducted on the structural properties and biological functions of the syndecan-4 cytoplasmic domain or extracellular domain. However, previous attempts to understand the structure and function of the syndecan-4 transmembrane domain (Syd4-TM) have been marred by experimental adversities due to insufficient yields and low solubility. To investigate the structural and functional properties of Syd4-TM, an efficient method of preparing milligram quantities of the corresponding peptide is necessary. Herein, we report an efficient method for the recombinant expression and purification of Syd4-TM peptide. Peptide was released from the fusion protein, and then purified by semi-preparative reversed-phase HPLC. Finally, more than 5mg of Syd4-TM peptide was obtained with high purity from 1l of M9 minimal media under optimized conditions. The preliminary biophysical properties of recombinant Syd4-TM peptide were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. The analysis of the CD spectrum shows that Syd4-TM adopts a stable α-helical structure in micelle environments. And solution NMR studies showed that Syd4-TM forms an asymmetric dimer in micelles.

Reductive Cyclotrimerization of Carbon Monoxide to the Deltate Dianion by an Organometallic Uranium Complex

Despite the long history of the Fischer-Tropsch reaction, carbon monoxide has proven remarkably resistant to selective homologation under mild conditions. Here, we find that an organouranium(III) complex induces efficient reductive trimerization of carbon monoxide at room temperature and pressure. The result is a triangular, cyclic C3O(2-)3, or deltate, dianion held between two uranium(IV) units. The bonding within the C3O(2-)3 unit and its coordination to the two U centers have been analyzed by x-ray diffraction and density functional theory computational studies, which show a stabilizing C-C agostic interaction between the C3 core and one U center. Solution nuclear magnetic resonance studies reveal a rapid equilibration of the deltate unit between the U centers.

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 β-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.