High-resolution NMR spectroscopy of membrane proteins in aligned bicelles

High-resolution solid-state NMR spectra can be obtained from uniformly (15)N-labeled membrane proteins in magnetically aligned bicelles. Fast uniaxial diffusion about the axis of the bilayer normal results in single-line spectra that contain the orientational information necessary for protein structure determination.     

Structure determination of a membrane protein with two trans-membrane helices in aligned phospholipid bicelles by solid-state NMR spectroscopy

The structure of the membrane protein MerFt was determined in magnetically aligned phospholipid bicelles by solid-state NMR spectroscopy. With two trans-membrane helices and a 10-residue inter-helical loop, this truncated construct of the mercury transport membrane protein MerF has sufficient structural complexity to demonstrate the feasibility of determining the structures of polytopic membrane proteins in their native phospholipid bilayer environment under physiological conditions. PISEMA, SAMMY, and other double-resonance experiments were applied to uniformly and selectively (15)N-labeled samples to resolve and assign the backbone amide resonances and to measure the associated (15)N chemical shift and (1)H-(15)N heteronuclear dipolar coupling frequencies as orientation constraints for structure calculations. (1)H/(13)C/(15)N triple-resonance experiments were applied to selectively (13)C'- and (15)N-labeled samples to complete the resonance assignments, especially for residues in the nonhelical regions of the protein. A single resonance is observed for each labeled site in one- and two-dimensional spectra. Therefore, each residue has a unique conformation, and all protein molecules in the sample have the same three-dimensional structure and are oriented identically in planar phospholipid bilayers. Combined with the absence of significant intensity near the isotropic resonance frequency, this demonstrates that the entire protein, including the loop and terminal regions, has a well-defined, stable structure in phospholipid bilayers.     

Structure determination of bioactive galloyl derivatives by NMR spectroscopy

The investigation of the chemical constituents of Symplocos racemosa Roxb led to the isolation of two new glycosides, symcomoside A (1) and symcomoside B (2), together with one known glycoside, tortoside C (3), which is reported for the first time from this plant. The structures of the new compounds were determined by 1D and 2D homonuclear and heteronuclear NMR spectroscopy, from chemical evidence and by comparison with published data for closely related compounds. Symcomoside B (2) showed potent inhibitory activity against alpha-glucosidase in a concentration-dependent fashion with an IC50 value of 0.733 +/- 0.033 mM whereas symcomoside A (1) showed very weak inhibitory activity against alpha-glucosidase (9.90% in 0.70 mM).     

Site-directed parallel spin-labeling and paramagnetic relaxation enhancement in structure determination of membrane proteins by solution NMR spectroscopy

A major challenge for the structure determination of integral membrane proteins by solution NMR spectroscopy is the limited number of NOE restraints in these systems stemming from extensive deuteration. Paramagnetic relaxation enhancement (PRE) by means of nitroxide spin-labels can provide valuable long-range distance information but, in practice, has limits in its application to membrane proteins because spin-labels are often incompletely reduced in highly apolar environments. Using the integral membrane protein OmpA as a model system, we introduce a method of parallel spin-labeling with paramagnetic and diamagnetic labels and show that distances in the range 15-24 Angstroms can be readily determined. The protein was labeled at 11 water-exposed and lipid-covered sites, and 320 PRE distance restraints were measured. The addition of these restraints resulted in significant improvement of the calculated backbone structure of OmpA. Structures of reasonable quality can even be calculated with PRE distance restraints only, i.e., in the absence of NOE distance restraints.     

Structure determination of ramosine, a guaianolide, by NMR spectroscopy

Ramosine, a new sesquiterpene lactone, was isolated from the chloroform fraction of Amberboa ramosa and the structure was assigned as 4beta-(hydroxymethyl)-3beta,4alpha-dihydroxy-8alpha-[(S)-3-hydroxy-2-ethylenepropionyloxy]-1alphaH, 5alphaH,6betaH,7alphaH,11betaH,11alpha-methylguaia-10(14)-en-6, 12-olide by extensive NMR studies.     

Tailoring 13C labeling for triple-resonance solid-state NMR experiments on aligned samples of proteins

In order to develop triple-resonance solid-state NMR spectroscopy of membrane proteins, we have implemented several different (13)C labeling schemes with the purpose of overcoming the interfering effects of (13)C-(13)C dipole-dipole couplings in stationary samples. The membrane-bound form of the major coat protein of the filamentous bacteriophage Pf1 was used as an example of a well-characterized helical membrane protein. Aligned protein samples randomly enriched to 35% (13)C in all sites and metabolically labeled from bacterial growth on media containing [2-(13)C]-glycerol or [1,3-(13)C]-glycerol enables direct (13)C detection in solid-state NMR experiments without the need for homonuclear (13)C-(13)C dipole-dipole decoupling. The (13)C-detected NMR spectra of Pf1 coat protein show a substantial increase in sensitivity compared to the equivalent (15)N-detected spectra. The isotopic labeling pattern was analyzed for [2-(13)C]-glycerol and [1,3-(13)C]-glycerol as metabolic precursors by solution-state NMR of micelle samples. Polarization inversion spin exchange at the magic angle (PISEMA) and other solid-state NMR experiments work well on 35% random fractionally and metabolically tailored (13)C-labeled samples, in contrast to their failure with conventional 100% uniformly (13)C-labeled samples.     

Structure, topology, and dynamics of membrane peptides and proteins from solid-state NMR spectroscopy

The high-resolution structure of membrane proteins is notoriously difficult to determine due to the hydrophobic nature of the protein-membrane complexes. Solid-state NMR spectroscopy is a unique and powerful atomic-resolution probe of the structure and dynamics of these important biological molecules. A number of new solid-state NMR methods for determining the depth of insertion, orientation, oligomeric structure, and long-range (10-15 A) distances of membrane proteins are summarized. Membrane protein depths can now be determined using several complementary techniques with varying site-specificity, distance precision, and mobility requirement on the protein. Membrane protein orientation can now be determined with or without macroscopic alignment, the latter providing a novel alternative for orientation determination of intrinsically curvature-inducing proteins. The novel analyses of beta-sheet membrane protein orientation are described. The quaternary structure of membrane peptide assemblies can now be elucidated using a 19F spin diffusion technique that simultaneously yields the oligomeric number and intermolecular distances up to 15 A. Finally, long-range distances up to approximately 10 A can now be measured using 1H spins with an accuracy of better than 1 A. These methods are demonstrated on several beta-sheet membrane peptides with antimicrobial activities and on two alpha-helical ion-channel proteins. Finally, we show that the nearly ubiquitous dynamics of membrane proteins can be readily examined using 2D correlation experiments. An intimate appreciation of molecular motion in these systems not only leads to important insights into the specific function of these membrane proteins but also may be exploited for other purposes such as orientation determination.     

Structure determination of selaginellins G and H from Selaginella pulvinata by NMR spectroscopy

Selaginellins G ( 1 ) and H ( 2 ), two new selaginellin derivatives, were isolated from the whole plant of Selaginella pulvinata. Their structures were elucidated, and complete assignments of the ¹H and ¹³C NMR spectroscopic data were achieved by 1D and 2D NMR experiments (HSQC, HMBC, COSY and ROESY). Compound 1 displayed good antifungal activity against Candida albicans with an IC₅₀ value of 5.3 µg/ml. Copyright © 2010 John Wiley & Sons, Ltd.     

High-resolution NMR spectroscopy of a GPCR in aligned bicelles

Solid-state NMR spectra with single-site resolution of CXCR1, a G protein-coupled receptor (GPCR), were obtained in magnetically aligned phospholipid bicelles. These results demonstrate that GPCRs in phospholipid bilayers are suitable samples for structure determination by solid-state NMR. The spectra also enable studies of drug-receptor interactions.     

Selective interface detection: mapping binding site contacts in membrane proteins by NMR spectroscopy

Intermolecular contact surfaces are important regions where specific interactions mediate biological function. We introduce a new magic angle spinning solid state NMR technique, dubbed "selective interface detection spectroscopy" (SIDY). In this technique, 13C-attached protons (1Hlig) are dephased by 1H-13C REDOR. A spin diffusion period is then used to enhance long distance 1H-1H correlations, and the results are detected by a short period of cross polarization to the 13C isotope labels. This SIDY approach allows selective observation of the interface between 13C-labeled and unlabeled moieties. We have used SIDY to probe the ligand-protein binding surface between a uniformly isotopically labeled ligand cofactor, U-13C20-11-cis-retinal, and its binding site in rhodopsin (Rho), an unlabeled, membrane-embedded G-protein coupled receptor (GPCR). The observed 1HGPCR-13Clig correlations indicate multiple close contacts between the protein and the ionone ring of the ligand, in agreement with binding studies. The polyene tail of the ligand displays fewer strong correlations in the SIDY spectrum. Some correlations can be assigned to the protein side chains lining the ligand binding site, in agreement with the crystal structure.     

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.     

Structure determination of di-O-benzylidene derivatives of L-iditol by 1H and 13C NMR spectroscopy

The configuration and stable conformation of two dibenzylidene-L -iditol isomers formed upon benzalation of L -iditol, as well as that of a third isomer obtained by partial hydrolysis of the tribenzylidene derivative—the main product of the benzalation reaction—were determined by ¹H and ¹³C NMR spectroscopy.     

Geometry determination of tetrasubstituted stilbenes by proton NMR spectroscopy

A simple spectroscopic method was applied to determine the geometry of tetrasubstituted alkenes. The observation of the ⁵J-coupling constants in proton NMR spectra on the ¹³C satellite signals could confirm the previous misassignment of 2,3-diphenylbutene. Hence, the (E)-isomer showed a 1.5 Hz coupling constant, whereas the (Z)-isomer showed a 1.1 Hz coupling constant. Based on this new assignment and a stereospecific preparation, we also propose a revision concerning the NMR data of 2,3-diphenyl-2,3-butanediol.     

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.     

Selenium determination in biological samples by X-ray emission spectroscopy

Determination of Se in blood serum by PIXE and XRF is presented. Two different sample preparation methods combined with two modes of sample excitation are compared. Both methods are shown to be suitable for Se determination in blood serum and in standard reference materials (horse kidney IAEA H-8 and bovine liver NBS 1577a).     

Structure determination of salvadorin, a novel dimeric dihydroisocoumarin from Salvadora oleoides, by NMR spectroscopy

Salvadorin, a new dimeric dihydroisocoumarin (1), was isolated from the chloroform fraction of Salvadora oleoides. Its chemical structure was established as 8-benzyl-6-[6-(6-ethyl-7-methyl-5,8-dihydro-2-naphthalenyl)-1-oxo-3,4-dihydro-1H-isochromen-8yl]-3, 4-dihyro-1H-isochromen-1-one, through spectroscopic techniques and chemical analysis.     

Structure determination of ursene-type triterpenes by NMR techniques

Two new ursene-type triterpenes, nudicauline A and nudicauline B, have been isolated from Launaea nudicaulis. Their structures have been assigned as 3beta-hydroxy-urs-11-ene (1) and 3beta-acetyl-urs-11-ene (2), respectively, by extensive NMR studies. In addition, olean-11,13(18)-diene (3), 3beta-hydroxy-13(28)-epoxy-urs-11-ene (4) and 3-keto-13(28)-epoxy-urs-11-ene (5) are also reported for the first time from this species.     

Nitrogen-14 solid-state NMR spectroscopy of aligned phospholipid bilayers to probe peptide-lipid interaction and oligomerization of membrane associated peptides

Characterization of the oligomerization of membrane-associated peptides is important to understand the folding and function of biomolecules like antimicrobial peptides, fusion peptides, amyloid peptides, toxins, and ion channels. However, this has been considered to be very difficult, because the amphipathic properties of the constituents of the cell membrane pose tremendous challenges to most commonly used biophysical techniques. In this study, we present the application of a simple (14)N solid-state NMR spectroscopy of aligned model membranes containing a phosphatidyl choline lipid to investigate the oligomerization of membrane-associated peptides. Since the near-symmetric nature of the choline headgroup of a phosphocholine lipid considerably reduces the (14)N quadrupole coupling, there are significant practical advantages in using (14)N solid-state NMR experiments to probe the interaction of peptide or protein with the surface of model membranes. Experimental results for several membrane-associated peptides are presented in this paper. Our results suggest that the experimentally measured (14)N quadrupole splitting of the lipid depends on the peptide-induced changes in the electrostatic potential of the lipid bilayer surface and therefore on the nature of the peptide, peptide-membrane interaction, and peptide-peptide interaction. It is inferred that the membrane orientation and oligomerization of the membrane-associated peptides can be measured using (14)N solid-state NMR spectroscopy.     

Structural determination of diterpenes from Daphne genkwa by NMR spectroscopy

Five daphnane type diterpenes have been isolated from the chloroform soluble fraction of Daphne genkwa. The structure of the new compound (1) was assigned as 5beta-hydroxyresiniferonol-6alpha,7alpha-epoxy-12beta-acetoxy-9,13,14-ortho-2E-decenoate by extensive NMR studies.