DHPC Micelles Research Articles

Peptide-bicelle interaction: Following variations in size and morphology by a combined NMR-SAXS approach

Changes in membrane properties occurring upon protein interaction are key questions in understanding membrane protein function. To report on the occurring size and shape variation we present here a combined NMR-SAXS method performed under physiological conditions using the same samples, enabling determination of a global parameter, the hydration radius (rH) and estimating the bicelle shape. We use zwitterionic (DMPC/DHPC) and negatively charged (DMPC/DHPC/DMPG) bicelles and investigate the interaction with model transmembrane and surface active peptides (KALP23 and melittin). 1H NMR measurements based mostly on the translational diffusion coefficient D determination are used to characterize cmc values of DHPC micelles under the investigated conditions, to describe DHPC distribution with exact determination of the q (long chain/short chain) lipid ratio, to estimate aggregation numbers and effective rH values. The scattering curve is used to fit a lenticular core-shell model enabling us to describe the bicelle shape in terms of ellipsoidal axis length parameters. For all studied systems formation of oblate ellipsoids is found. Even though the rG/rH ratio would be an elegant way to characterize shape variations, we show that changes occurring upon peptide-bicelle interaction in the “effective” size and in the measure on the anisometry - morphology - of the objects can be described by using rH and the simplistic ellipsoidal core-shell model. While the influence of the transmembrane KALP peptide is significant, effects upon addition of surface active melittin peptide seem negligible. This synergy of techniques under controlled conditions can provide information about bicellar shape modulation occurring during peptide-bicelle interactions.

Molecular Interactions of Lipopolysaccharide with an Outer Membrane Protein from Pseudomonas aeruginosa Probed by Solution NMR.

Pseudomonas aeruginosa is an opportunistic human pathogen causing pneumonias that are particularly severe in cystic fibrosis and immunocompromised patients. The outer membrane (OM) of P. aeruginosa is much less permeable to nutrients and other chemical compounds than that of Escherichia coli. The low permeability of the OM, which also contributes to Pseudomonas' significant antibiotic resistance, is augmented by the presence of the outer membrane protein H (OprH). OprH directly interacts with lipopolysaccharides (LPS) that constitute the outer leaflet of the OM and thus contributes to the structural stability of the OM. In this study, we used solution NMR spectroscopy to characterize the interactions between LPS and OprH in molecular detail. NMR chemical shift perturbations observed upon the addition of LPS to OprH in DHPC micelles indicate that this interaction is predominantly electrostatic and localized to the extracellular loops 2 and 3 and a number of highly conserved basic residues near the extracellular barrel rim of OprH. Single-site mutations of these residues were not enough to completely abolish binding, but OprH with cumulative mutations of Lys70, Arg72, and Lys103 no longer binds LPS. The dissociation constant (∼200 μM) measured by NMR is sufficient to efficiently bind LPS to OprH in the OM. This work highlights that solution NMR is suitable to study specific interactions of lipids with integral membrane proteins and provides a detailed molecular model for the interaction of LPS with OprH; i.e., an interaction that contributes to the integrity of the OM of P. aeruginosa under low divalent cation and antibiotic stress conditions. These methods should thus be useful for screening antibiotics that might disrupt OprH-LPS interactions and thereby increase the permeability of the OM of P. aeruginosa.

Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilization in DDM, DDM/CHAPS, or DHPC micelles, although A2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. The studies presented in this paper also underline the importance of the protein’s purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.

NMR Solution Structure and Extracellular Loop Dynamics of the Outer Membrane Protein OprG of Pseudomonas Aeruginosa Explain Transport of Small Amino Acids

Pseudomonas aeruginosa is an opportunistic human pathogen that is responsible for a growing number of nosocomial infections and the main cause of death in patients with cystic fibrosis. The structure of one of its outer membrane proteins (OprG) has been solved by x-ray crystallography and revealed a tall 8-stranded β-barrel that extends far into the extracellular space with a lumen lined with hydrophobic residues (Touw et al, PLoS ONE 5:15016[2011]). An interesting feature of that structure is a lateral proline-rich opening in the barrel wall near the membrane interface that has been suggested to function as a gate for small hydrophobic molecules that might subsequently diffuse across the bilayer of the outer membrane.In order to test this hypothesis we re-determined the structure of OprG in DHPC micelles by NMR. We show that the β-barrel has substantially shorter strands than in the crystal structure and longer extracellular loops without a defined lateral gate. To further investigate the role of the prolines in the proposed gate, we mutated them to alanines and determined the structure of the P92A mutant. Introduction of the P92A mutation resulted in an asymmetric elongation of the beta-barrel and changed the loop dynamics and the hydrogen-bonding pattern in the barrel-to-loop transition regions. As shown on a separate poster by PS, IK, and LKT, the physiological substrates of OprG are small amino acids and the P92A mutation aborts small amino acid transport.

An extended combinatorial 15N, 13Cα, and 13C' labeling approach to protein backbone resonance assignment.

Solution NMR studies of α-helical membrane proteins are often complicated by severe spectral crowding. In addition, hydrophobic environments like detergent micelles, isotropic bicelles or nanodiscs lead to considerably reduced molecular tumbling rates which translates into line-broadening and low sensitivity. Both difficulties can be addressed by selective isotope labeling methods. In this publication, we propose a combinatorial protocol that utilizes four different classes of labeled amino acids, in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in (15)N or (13)C isotopes individually as well as simultaneously. This results in eight different combinations of dipeptides giving rise to cross peaks in (1)H-(15)N correlated spectra. Their differentiation is achieved by recording a series of HN-detected 2D triple-resonance spectra. The utility of this new scheme is demonstrated with a homodimeric 142-residue membrane protein in DHPC micelles. Restricting the number of selectively labeled samples to three allowed the identification of the amino-acid type for 77 % and provided sequential information for 47 % of its residues. This enabled us to complete the backbone resonance assignment of the uniformly labeled protein merely with the help of a 3D HNCA spectrum, which can be collected with reasonable sensitivity even for relatively large, non-deuterated proteins.

Prolines Around Hypothetical Active Site are Important for the Structure and Dynamics of the Outer Membrane Protein G from Pseudomonas Aeruginosa

The outer membrane protein G (OprG) from Pseudomonas aeruginosa is a beta-barrel protein hypothesized to be responsible for the uptake of hydrophobic compounds. A crystal structure at 2.4 A resolution reveals an 8-stranded beta-barrel with four long extracellular loops and a lumen lined almost exclusively with hydrophobic residues. Another interesting feature is a lateral opening in the barrel wall, surrounded by three proline residues (P66, P91, P92), which has been suggested to mediate diffusion of small hydrophobic molecules across the outer membrane by a mechanism similar to that of E. coli FadL protein (Touw et al (2011), PLoS ONE 5(11):15016, van den Berg et al (2004), Science 304:1506-1509). We determined the NMR structure of OprG in DHPC micelles at pH 6.0, which shows the beta-barrel region to have substantially shorter strands and longer periplasmic loops than the crystal structure. To further investigate the properties of OprG, we mutated three prolines around the hypothetical active site to alanines and collected 15N-1H TROSY spectra of the mutants. The P92A mutant showed high quality spectra and was used for structure determination. Introducing the P92A mutation results in an asymmetric elongation of the beta-barrel region and changed the hydrogen bonding network and loops dynamics. Although the proline mutants incorporate into membranes less efficiently than the wild-type, our single molecule tracking experiments and fluorescence measurements indicate that they all form multimers of varying sizes in membranes. This study shows the importance of the prolines around the hypothetical active site for the OprG structure and their impact on the protein's properties in membranes.

Lipid Transfer Kinetics from Nanolipoprotein Particles to Bicelles

Nanolipoprotein particles (NLPs), also known as nanodiscs, are lipid bilayers bounded by apolipoprotein. We recently showed that lipids and membrane proteins cannot exchange between NLPs. However, addition of bicelles opens NLPs and transfers their contents to bicelles, which freely exchange lipids and proteins. Because monomeric membrane proteins can be prepared in NLPs by cell-free protein synthesis, the bicelle-induced transfer process may provide a new method for studying membrane protein oligomerization. The mechanism of the NLP-bicelle interaction is unknown. We have now tested the effects of bicelle detergent (DHPC), apolipoprotein (MSP1E3D1), and temperature on lipid transfer from NLPs to bicelles, using stopped-flow kinetics. NLPs were prepared with fluorescent lipids (0.02 to 0.05 mole fraction), consisting of FRET donors (NBD-PE) and acceptors (LR-PE) at approximately equal concentrations. NLPs were mixed with a 200-fold molar excess of DHPC/DMPC bicelles (equimolar 6- and 14-carbon acyl chains) in a stopped-flow fluorometer. The rate of lipid transfer was monitored by the appearance of unquenched NBD fluorescence at 520 nm. The observed pseudo-first-order rate constant was surprisingly small (0.26/sec). NLPs did not react with DHPC alone below its critical micelle concentration (cmc). Above the cmc, the reaction was complete within the instrument dead time. Thus, the rate-limiting step is not the reaction of NLPs with DHPC monomers or micelles. Added MSP1E3D1 had no effect on the rate, ruling out free apolipoprotein involvement. The NLP-bicelle mixing rate showed a strong temperature dependence (activation energy = 28 kcal/mol). Near or below the DMPC phase transition temperature, the kinetics were biphasic. The results suggest NLP-bicelle mixing kinetics may be mechanistically similar to lipid mixing via fusion pores.

Resonance assignments and secondary structure of apolipoprotein E C-terminal domain in DHPC micelles.

Human apolipoprotein E (apoE) has been known to play a key role in the transport of plasma cholesterol and lipoprotein metabolism. It is an apolipoprotein of 299 amino acids with a molecular mass, ~34kDa. ApoE has three major isoforms, apoE2, apoE3, and apoE4 which differ only at residue 112 or 158. ApoE consists of two independently folded domains (N-terminal and C-terminal domain) separated by a hinge region. The N-terminal domain and C-terminal domain of apoE are responsible for the binding to receptor and to lipid, respectively. Since the high resolution structures of apoE in lipids are still unavailable to date, we therefore aim to resolve the structures in lipids by NMR. Here, we reported the resonance assignments and secondary structure distribution of the C-terminal domain of wild-type human apoE (residue 195-299) in the micelles formed by dihexanoylphosphatidylcholine. Our results may provide a novel structural model of apoE in micelles and may shed new light on the molecular mechanisms underlying the apoE related biological processes.

NMR Analyses of the Structure and Dynamics of Klebsiella Pneumoniae OMPA Domains and Full Length Protein

Collaborations:Andreas Engel and Daniel Muller, ETH Zurich, Basel, Switzerland.G. Pintacuda, CRMN, UMR5280, Villeurbanne, France.The transmembrane domain of kpOmpA possesses four long extracellular loops which exhibit substantial sequence variability throughout OmpA homologues in Enterobacteria. These loops are responsible for the immunological properties of the protein, such as cellular and humoral recognitions. Using liquid state NMR we have determined the 3D structure of kpOmpA in DHPC micelles (M. Renault et al., J. Mol. Biol. 2009). In a micellar environment, a complex dynamical behavior has been observed: a rigid barrel core, ms motion at the micellar-water interface, and sub-ns motion within the loops. Using solid state NMR relaxation and proteolysis experiments, we have demonstrated the persistence of this complex motional behavior in E. coli polar lipid bilayers (I. Iordanov et al., Biochim. Biophys. Acta, 2012). Using single molecule force spectroscopy (with D. Muller and A. Engel) we have shown that kpOmpA is able to unfold and refold reversibly its β-barrel core (P. Bosshart et al., Structure 2012).Recent advances involve: a) characterizing the structure of its C-terminal domain and its interaction with the peptido-glycane; b) analyzing ssNMR spectra of N-terminal membrane domain in liposomes using MAS at 1 GHz and 60 kHz spinning frequency (with G. Pintacuda); c) comparing the NMR spectra of the various domains and the full length protein in solution, in liposomes and in intact cell envelopes using cellular solid state NMR as established in (M. Renault et al., PNAS 2012).

The Structure and Oligomericity of the Transmembrane Domain of Cytokine Receptors is Modulated by the Protein/Lipid Ratio

The prolactin receptor (PRLR) and the growth hormone receptor (GHR) are archetypical members of the class-I-hematopoietic-cytokine receptor superfamily. They are single-pass transmembrane receptors operating as dimers in the cell membrane. Receptors from this family are involved in many key processes, and linked to numerous pathologies and cancers. Currently, the cross-membrane signal transduction through these receptors is an enigma; no structures of any class-I-cytokine receptor transmembrane domain (TMD) has been solved, nor has a general mechanism been proposed to account for the propagation of the hormone binding signal. Deciphering the signaling mechanism and structural details of the TMD is a prerequisite for understanding these receptors and for development of agonists and antagonists.With nuclear magnetic resonance (NMR) spectroscopy as the main tool, we have investigated the structural characteristics of GHR-TMD and PRLR-TMD and the influence of the solvent in which they are embedded. We have recorded heteronuclear, multidimensional NMR spectra of the TMDs in different membrane mimetics (micelles,bicelles,amphipols) of different lipid composition. We have acquired residue-specific structural information on PRLR-TMD and confirmed the predicted alpha-helical secondary structure. Surprisingly, it is found that the structural characteristics of PRLR-TMD embedded in DHPC micelles and POPC/POPS bicelles are equivalent. We observe for both TMDs that the amount of available lipid solvent rather than the membrane mimetics and lipid composition, perturbs the structural physiognomies of the membrane-embedded domains. We hypothesize that these perturbations relates to the TMD dimerization. The NMR data combined with chemical cross-linking suggest a weak intrinsic propensity of the TMD for dimerization, and that entropic forces exerted by the surrounding lipids therefore may be crucial to drive the TMD dimerization and activation. These findings encourage further investigations of the functional significance of the receptor-membrane interplay.

Peptaibol Antiamoebin I: Spatial Structure, Backbone Dynamics, Interaction with Bicelles and Lipid‐Protein Nanodiscs, and Pore Formation in Context of Barrel‐Stave Model

Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. In comparison with other 16-amino acid-residue peptaibols, e.g., zervamicin IIB (Zrv-IIB), Aam-I possesses relatively weak biological and channel-forming activities. In MeOH solution, Aam-I demonstrates fast cooperative transitions between right-handed and left-handed helical conformation of the N-terminal (1-8) region. We studied Aam-I spatial structure and backbone dynamics in the membrane-mimicking environment (DMPC/DHPC bicelles)(1) ) by heteronuclear (1) H,(13) C,(15) N-NMR spectroscopy. Interaction with the bicelles stabilizes the Aam-I right-handed helical conformation retaining significant intramolecular mobility on the ms-μs time scale. Extensive ms-μs dynamics were also detected in the DPC and DHPC micelles and DOPG nanodiscs. In contrast, Zrv-IIB in the DPC micelles demonstrates appreciably lesser mobility on the μs-ms time scale. Titration with Mn(2+) and 16-doxylstearate paramagnetic probes revealed Aam-I binding to the bicelle surface with the N-terminus slightly immersed into hydrocarbon region. Fluctuations of the Aam-I helix between surface-bound and transmembrane (TM) state were observed in the nanodisc membranes formed from the short-chain (diC12 : 0) DLPC/DLPG lipids. All the obtained experimental data are in agreement with the barrel-stave model of TM pore formation, similarly to the mechanism proposed for Zrv-IIB and other peptaibols. The observed extensive intramolecular dynamics explains the relatively low activity of Aam-I.

Modeling the Self-Assembly and Stability of DHPC Micelles Using Atomic Resolution and Coarse Grained MD Simulations.

Membrane mimics such as micelles and bicelles are widely used in experiments involving membrane proteins. With the aim of being able to carry out molecular dynamics simulations in environments comparable to experimental conditions, we set out to test the ability of both coarse grained and atomistic resolution force fields to model the experimentally observed behavior of the lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), which is a widely used lipid for biophysical characterization of membrane proteins. It becomes clear from our results that a satisfactory modeling of DHPC aggregates in solution poses different demands to the force field than do the modeling of bilayers. First, the representation of the short tailed lipid DHPC in the coarse grained force field MARTINI is assessed with the intend of successfully self-assemble micelles with structural characteristics comparable to experimental data. Then, the use of the recently presented polarizable water model in MARTINI is shown to be essential for producing micelles that are structurally in accordance with experiments. For the atomistic representations of DHPC micelles in solution the GROMOS96 force field with lipid parameters by A. Kukol fails to maintain stable micelles, whereas the most recent CHARMM36 lipid parameters and GROMOS96 with the so-called Berger lipid parameters both succeed in this regard.

Secondary structure, dynamics, and architecture of the p7 membrane protein from hepatitis C virus by NMR spectroscopy

P7 is a small membrane protein that is essential for the infectivity of hepatitis C virus. Solution-state NMR experiments on p7 in DHPC micelles, including hydrogen/deuterium exchange, paramagnetic relaxation enhancement and bicelle ‘q-titration,’ demonstrate that the protein has a range of dynamic properties and distinct structural segments. These data along with residual dipolar couplings yield a secondary structure model of p7. We were able to confirm previous proposals that the protein has two transmembrane segments with a short interhelical loop containing the two basic residues K33 and R35. The 63-amino acid protein has a remarkably complex structure made up of seven identifiable sections, four of which are helical segments with different tilt angles and dynamics. A solid-state NMR two-dimensional separated local field spectrum of p7 aligned in phospholipid bilayers provided the tilt angles of two of these segments. A preliminary structural model of p7 derived from these NMR data is presented.

Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K + channels

The membrane location of two fragments in two different K +-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative “paddle” domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q = 0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T 1 and 13C– 1H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons. 2H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems.

NMR Structural Studies of Interactions of a Small, Nonpeptidyl Tpo Mimic with the Thrombopoietin Receptor Extracellular Juxtamembrane and Transmembrane Domains

Thrombopoietin (Tpo) is a glycoprotein growth factor that supports hematopoietic stem cell survival and expansion and is the principal regulator of megakaryocyte growth and differentiation. Several small, nonpeptidyl molecules have been identified as selective human Tpo receptor (hTpoR) agonists. To understand how the small molecule Tpo mimic SB394725 interacts and activates hTpoR, we performed receptor domain swap and mutagenesis studies. The results suggest that SB394725 interacts specifically with the extracellular juxtamembrane region (JMR) and the transmembrane (TM) domain of hTpoR. Solution and solid-state NMR structural studies using a peptide containing the JMR-TM sequences showed that this region of hTpoR, unexpectedly, consists of two alpha-helices separated by a few nonhelical residues. SB394725 interacts specifically with His-499 in the TM domain and a few distinct residues in the JMR-TM region and affects several specific C-terminal TM domain residues. The unique structural information provided by these studies both sheds light on the distinctive mechanism of action of SB394725 and provides valuable insight into the mechanism of ligand-induced cytokine receptor activation.

NMR Solution Structure of the Peptide Fragment 1−30, Derived from Unprocessed Mouse Doppel Protein, in DHPC Micelles

The downstream prion-like Doppel (Dpl) protein is a homologue related to the prion protein (PrP). Dpl is expressed in the brains of mice that do not express PrP, and Dpl is known to be toxic to neurons. One mode of toxicity has been suggested to involve direct membrane interactions. PrP under certain conditions of cell trafficking retains an uncleaved signal peptide, which may also hold for the much less studied Dpl. For a peptide with a sequence derived from the N-terminal part (1-30) of mouse Dpl (mDpl(1-30)) CD spectroscopy shows about 40% alpha-helical structure in DHPC and SDS micelles. In aqueous solution it is mostly a random coil. The three-dimensional solution structure was determined by NMR for mDpl(1-30) associated with DHPC micelles. 2D 1H NMR spectra of the peptide in q = 0.25 DMPC/DHPC bicelles only showed signals from the unstructured termini, indicating that the structured part of the peptide resides within the lipid bilayer. Together with 2H2O exchange data in the DHPC micelle solvent, these results show an alpha-helix protected from solvent exchange between residues 7 and 19, and suggest that the alpha-helical segment can adopt a transmembrane localization also in a membrane. Leakage studies with entrapped calcein in large unilamellar phospholipid vesicles showed that the peptide is almost as membrane perturbing as melittin, known to form pores in membranes. The results suggest a possible channel formation mechanism for the unprocessed Dpl protein, which may be related to toxicity through direct cell membrane interaction and damage.

NMR Solution Structure and Membrane Interaction of the N-Terminal Sequence (1−30) of the Bovine Prion Protein

The structure and membrane interaction of the N-terminal sequence (1-30) of the bovine prion protein (bPrPp) has been investigated by NMR spectroscopy in phospholipid membrane mimetic systems. CD spectroscopy revealed that the peptide adopts a largely alpha-helical structure in zwitterionic bicelles as well as in DHPC micelles but has a less degree of alpha-helix structure in partly charged bicelles. The solution structure of bPrPp was determined in DHPC micelles, and an alpha-helix was found between residues Ser8 and Ile21. The residues within the helical region show slow amide hydrogen exchange. Translational diffusion measurements in zwitterionic q = 0.5 bicelles show that the peptide does not induce aggregation of the bicelles. Increased quadrupolar splittings were observed in the outer part of the (2)H spectrum of DMPC in q = 3.5 bicelles, indicating that the peptide induces a certain degree of order in the bilayer. The amide hydrogen exchange and the (2)H NMR results are consistent with a slight positive hydrophobic mismatch and that bPrPp forms a stable helix that inserts in a transmembrane location in the bilayer. The structure of bPrPp and its position in the membrane may be relevant for the understanding of how the N-terminal (1-30) part of the bovine PrP functions as a cell-penetrating peptide. These findings may lead to a better understanding of how the prion protein accumulates at the membrane surface and also how the conversion into the scrapie form is carried out.

NMR Structure of the Integral Membrane Protein OmpX

The structure of the integral membrane protein OmpX from Escherichia coli reconstituted in 60 kDa DHPC micelles (OmpX/DHPC) was calculated from 526 NOE upper limit distance constraints. The structure determination was based on complete sequence-specific assignments for the amide protons and the Val, Leu, and Ile(δ 1) methyl groups in OmpX, which were selectively protonated on a perdeuterated background. The solution structure of OmpX in the DHPC micelles consists of a well-defined, eight-stranded antiparallel β-barrel, with successive pairs of β-strands connected by mobile loops. Several long-range NOEs observed outside of the transmembrane barrel characterize an extension of a four-stranded β-sheet beyond the height of the barrel. This protruding β-sheet is believed to be involved in intermolecular interactions responsible for the biological functions of OmpX. The present approach for de novo structure determination should be quite widely applicable to membrane proteins reconstituted in mixed micelles with overall molecular masses up to about 100 kDa, and may also provide a platform for additional functional studies.

Stereospecific assignments of the isopropyl methyl groups of the membrane protein OmpX in DHPC micelles.

In NMR studies of large molecular structures, the number of conformational constraints based on NOE measurements is typically limited due to the need for partial deuteration. As a consequence, when using selective protonation of peripheral methyl groups on a perdeuterated background, stereospecific assignments of the diastereotopic methyl groups of Val and Leu can have a particularly large impact on the quality of the NMR structure determination. For example, 3D 15N- and 13C-resolved [1H,1H]-NOESY spectra of the E. Coli membrane protein OmpX in mixed micelles with DHPC, which have an overall molecular weight of about 60 kDa, showed that about 50% of all obtainable NOEs involve the diastereotopic methyl groups of Val and Leu. In this paper, we used biosynthetically-directed fractional 13C labeling of OmpX and [13C,1H]-HSQC spectroscopy to obtain stereospecific methyl assignments of Val and Leu in OmpX/DHPC. For practical purposes it is of interest that this data could be obtained without use of a deuterated background, and that combinations of NMR experiments have been found for obtaining the desired information either at a 1H frequency of 500 MHz, or with significantly reduced measuring time on a high-frequency instrument.

Side chain NMR assignments in the membrane protein OmpX reconstituted in DHPC micelles.

Sequence-specific assignments have been obtained for side chain methyl resonances of Val, Leu and Ile in the outer membrane protein X (OmpX) from Escherichia coli reconstituted in 60 kDa micelles in aqueous solution. Using previously established techniques, OmpX was uniformly 2H,13C,15N-labeled with selectively protonated Val-gamma(1,2), Leu-delta(1,2) and Ile-delta1 methyl groups. The thus labeled protein was studied with the novel experiments 3D (H)C(CC)-TOCSY-(CO)-[15N,1H]-TROSY and 3D H(C)(CC)-TOCSY-(CO)-[15N,1H]-TROSY. Compared to the corresponding conventional experimental schemes, the TROSY-type experiments yielded a sensitivity gain of about 2 at 500 MHz. The overall sensitivity of the experiments was further enhanced more than two-fold by the use of a cryoprobe. Complete assignments of the proton and carbon chemical shifts were obtained for all isopropyl methyl groups of Val and Leu, as well as for the delta1-methyls of Ile. The present approach is applicable for soluble proteins or micelle-reconstituted membrane proteins in structures with overall molecular weights up to about 100 kDa, and adds to the potentialities of solution NMR for de novo structure determination as well as for functional studies, such as ligand screening with proteins in large structures.