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A study was conducted to characterize a two-dimensional nuclear magnetic resonance method for the measurement of dipolar couplings in polycrystalline solids. A home-built spectrometer was utilized to perform homonuclear two-dimensional DRAWS correlation studies. The phase of the initial 1H pulse for cross polariation was inverted between successive studies to hinder zero frequency peaks. Results indicated that a pulse sequence supporting a DRAWS irradiation for dipolar recoupling can effectively determine two dimensional spectra.

Interactions between 15N-labelled peptides or proteins and lipids can be investigated using membranes aligned on a thin polymer film, which is rolled into a cylinder and inserted into the MAS-NMR rotor. This can be spun at high speed, which is often useful at high field strengths. Unfortuantely, substrate films like commercially available polycarbonate or PEEK produce severe overlap with peptide and protein signals in 13C-MAOSS NMR spectra. We show that a simple house hold foil support allows clear observation of the carbonyl, aromatic and Cα signals of peptides and proteins as well as the ester carbonyl and choline signals of phosphocholine lipids. The utility of the new substrate is validated in applications to the membrane active peptide zervamicin IIB. The stability and macroscopic ordering of thin PC10 bilayers was compared with that of thicker POPC bilayers, both supported on the household foil. Sidebands in the 31P-spectra showed a high degree of alignment of both the supported POPC and PC10 lipid molecules. Compared with POPC, the PC10 lipids are slightly more disordered, most likely due to the increased mobilities of the shorter lipid molecules. This mobility prevents PC10 from forming stable vesicles for MAS studies. The 13C-peptide peaks were selectively detected in a 13C-detected 1H-spin diffusion experiment. Qualitative analysis of build-up curves obtained for different mixing times allowed the transmembrane peptide in PC10 to be distinguished from the surface bound topology in POPC. The 13C-MAOSS results thus independently confirms previous findings from 15N spectroscopy [Bechinger, B., Skladnev, D.A., Ogrel, A., Li, X., Rogozhkina, E.V., Ovchinnikova, T.V., O’Neil, J.D.J. and Raap, J. (2001) Biochemistry, 40, 9428–9437]. In summary, application of house hold foil opens the possibility of measuring high resolution 13C-NMR spectra of peptides and proteins in well ordered membranes, which are required to determine the secondary and supramolecular structures of membrane active peptides, proteins and aggregates. [ABSTRACT FROM AUTHOR]

Membrane fusion requires restructuring of lipid bilayers mediated by fusogenic membrane proteins. Peptides that correspond to natural transmembrane sequences or that have been designed to mimic them, such as low-complexity "Leu-Val" (LV) peptide sequences, can drive membrane fusion, presumably by disturbing the lipid bilayer structure. Here, we assess how peptides of different fusogenicity affect membrane structure using solid state NMR techniques. We find that the more fusogenic variants induce an unaligned lipid phase component and a large degree of phase separation as observed in (31)P 2D spectra. The data support the idea that fusogenic peptides accumulate PE in a non-bilayer phase which may be critical for the induction of fusion., (Copyright 2009 Elsevier B.V. All rights reserved.)

Membrane fusion requires drastic and transient changes of bilayer curvature and here we have studied the interaction of three de novo designed synthetic hydrophobic peptides with a biomimetic three-lipid mixture by solid state NMR. An experimental approach is presented for screening of peptide-lipid interactions and their aggregation, and their embedding in a biomimetic membrane system using established proton-decoupled 13C,15N and proton spin diffusion heteronuclear 1H-13C correlation NMR methods at high magnetic field. Experiments are presented for a set of de-novo designed fusion peptides in interaction with their lipid environment. The data provide additional support for the transmembrane model for the least fusogenic peptide, L16, while the peripheral intercalation model is preferred for the fusogenic peptides LV16 and LV16G8P9. This contributes to converging evidence that peripheral intercalation is both necessary and sufficient to trigger the fusion process for a lipid mixture close to a critical point for phase separation across the bilayer.

The electronic ground states of the bacteriochlorophyll a type B800 and type B850 in the light-harvesting 2 complex of Rhodopseudomonas acidophila strain 10050 have been characterized by magic angle spinning (MAS) dipolar (13)C-(13)C correlation NMR spectroscopy. Uniformly [(13)C,(15)N] enriched light-harvesting 2 (LH2) complexes were prepared biosynthetically, while [(13)C,(15)N]-B800 LH2 complexes were obtained after reconstitution of apoprotein with uniformly [(13)C,(15)N]-enriched bacteriochlorophyll cofactors. Extensive sets of isotropic (13)C NMR chemical shifts were obtained for each bacteriochlorin ring species in the LH2 protein. (13)C isotropic shifts in the protein have been compared to the corresponding shifts of monomeric BChl a dissolved in acetone-d(6). Density functional theory calculations were performed to estimate ring current effects induced by adjacent cofactors. By correction for the ring current shifts, the (13)C shift effects due to the interactions with the protein matrix were resolved. The chemical shift changes provide a clear evidence for a global electronic effect on the B800 and B850 macrocycles, which is attributed to the dielectrics of the protein environment, in contrast with local effects due to interaction with specific amino acid residues. Considerable shifts of -6.2 < Deltasigma < +5.8 ppm are detected for (13)C nuclei in both the B800 and the B850 bacteriochlorin rings. Because the shift effects for the B800 and B850 are similar, the polarization of the electronic ground states induced by the protein environment is comparable for both cofactors and corresponds with a red shift of approximately 30 nm relative to the monomeric BChl dissolved in acetone-d(6). The electronic coupling between the B850 cofactors due to macrocycle overlap is the predominant mechanism behind the additional red shift in the B850.

We have obtained carbon-carbon bond length data for the functional retinylidene chromophore of rhodopsin, with a spatial resolution of 3 pm. The very high resolution was obtained by performing double-quantum solid-state NMR on a set of noncrystalline isotopically labelled bovine rhodopsin samples. We detected localized perturbations of the carbon-carbon bond lengths of the retinylidene chromophore. The observations are consistent with a model in which the positive charge of the protonated Schiff base penetrates into the polyene chain and partially concentrates around the C13 position. This coincides with the proximity of a water molecule located between the glutamate-181 and serine-186 residues of the second extracellular loop, which is folded back into the transmembrane region. These measurements support the hypothesis that the polar residues of the second extracellular loop and the associated water molecule assist the rapid selective photoisomerization of the retinylidene chromophore by stabilizing a partial positive charge in the center of the polyene chain.

Reaction centers of wild-type Rhodobacter sphaeroides were selectively (13)C-isotope labeled in bacteriochlorophyll and bacteriopheophytin. (13)C solid-state CP/MAS NMR and photo-CIDNP were used to provide insight into the electronic structure of the primary electron donor and acceptor on the atomic scale. The first 2-dimensional photochemically induced dynamic nuclear polarization (photo-CIDNP) (13)C-(13)C solid-state MAS NMR spectra reveal that negative charging of the two BChl rings of the primary donor is involved in ground-state tuning of the oxidation potential of these cofactors in the protein via local electrostatic interactions. In particular, the (13)C shifts show moderate differences in the electronic structure between the two BChl molecules of the special pair in the electronic ground state, which can be attributed to hydrogen bonding of one of the BChl molecules. The major fraction of the electron spin density is strongly delocalized over the two BChl molecules of the special pair and the photochemically active BPhe. A small fraction of the pi-spin density is distributed over a fourth component, which is assigned to the accessory BChl. Comparison of the photo-CIDNP data with "dark" NMR spectra obtained in ultra high field indicates a rigid special pair environment upon photoreaction and suggests that structural changes of the aromatic macrocycles of the two BChl molecules of the special pair do not significantly contribute to the reorganization energy associated with the charge-transfer process.

Rhodopsin is a member of the superfamily of G-protein-coupled receptors. This seven alpha-helix transmembrane protein is the visual pigment of the vertebrate rod photoreceptor cells that mediate dim light vision. In the active binding site of this protein the ligand or chromophore, 11-cis-retinal, is covalently bound via a protonated Schiff base to lysine residue 296. Here we present the complete (1)H and (13)C assignments of the 11-cis-retinylidene chromophore in its ligand-binding site determined with ultra high field magic angle spinning NMR. Native bovine opsin was regenerated with 99% enriched uniformly (13)C-labeled 11-cis-retinal. From the labeled pigment, (13)C carbon chemical shifts could be obtained by using two-dimensional radio frequency-driven dipolar recoupling in a solid-state magic angle spinning homonuclear correlation experiment. The (1)H chemical shifts were assigned by two-dimensional heteronuclear ((1)H-(13)C) dipolar correlation spectroscopy with phase-modulated Lee-Goldburg homonuclear (1)H decoupling applied during the t(1) period. The data indicate nonbonding interactions between the protons of the methyl groups of the retinylidene ionone ring and the protein. These nonbonding interactions are attributed to nearby aromatic acid residues Phe-208, Phe-212, and Trp-265 that are in close contact with, respectively, H-16/H-17 and H-18. Furthermore, binding of the chromophore involves a chiral selection of the ring conformation, resulting in equatorial and axial positions for CH(3)-16 and CH(3)-17.

Proton spin-lattice relaxation rate constants have been measured as a function of magnetic field strength for water, water-glycerol solution, cyclohexane, methanol, benzene, acetone, acetonitrile, and dimethyl sulfoxide. The magnetic relaxation dispersion is well approximated by a Lorentzian shape. The origin of the relaxation dispersion is identified with the paramagnetic contribution from molecular oxygen. In the small molecule cases studied here, the effective correlation time for the electron-nuclear coupling may include contributions from both translational diffusion and the electron T(1). The electron T(1) for molecular oxygen dissolved in several solvents was found to be approximately 7.5 ps and nearly independent of solvent or viscosity., (Copyright 2001 Academic Press.)

Water proton spin-lattice relaxation is studied in dilute solutions of bovine serum albumin as a function of magnetic field strength, oxygen concentration, and solvent deuteration. In contrast to previous studies conducted at high protein concentrations, the observed relaxation dispersion is accurately Lorentzian with an effective correlation time of 41 +/- 3 ns when measured at low proton and low protein concentrations to minimize protein aggregation. Elimination of oxygen flattens the relaxation dispersion profile above the rotational inflection frequency, nearly eliminating the high field tail previously attributed to a distribution of exchange times for either whole water molecules or individual protons at the protein-water interface. The small high-field dispersion that remains is attributed to motion of the bound water molecules on the protein or to internal protein motions on a time scale of order one ns. Measurements as a function of isotope composition permit separation of intramolecular and intermolecular relaxation contributions. The magnitude of the intramolecular proton-proton relaxation rate constant is interpreted in terms of 25 +/- 4 water molecules that are bound rigidly to the protein for a time long compared with the rotational correlation time of 42 ns. This number of bound water molecules neglects the possibility of local motions of the water in the binding site; inclusion of these effects may increase the number of bound water molecules by 50%.

Proteins play an important role in the biological mechanisms controlling hard tissue development, but the details of molecular recognition at inorganic crystal interfaces remain poorly characterized. We have applied a recently developed homonuclear dipolar recoupling solid-state NMR technique, dipolar recoupling with a windowless sequence (DRAWS), to directly probe the conformation of an acidic peptide adsorbed to hydroxyapatite (HAP) crystals. The phosphorylated hexapeptide, DpSpSEEK (N6, where pS denotes phosphorylated serine), was derived from the N terminus of the salivary protein statherin. Constant-composition kinetic characterization demonstrated that, like the native statherin, this peptide inhibits the growth of HAP seed crystals when preadsorbed to the crystal surface. The DRAWS technique was used to measure the internuclear distance between two 13C labels at the carbonyl positions of the adjacent phosphoserine residues. Dipolar dephasing measured at short mixing times yielded a mean separation distance of 3.2 +/- 0.1 A. Data obtained by using longer mixing times suggest a broad distribution of conformations about this average distance. Using a more complex model with discrete alpha-helical and extended conformations did not yield a better fit to the data and was not consistent with chemical shift analysis. These results suggest that the peptide is predominantly in an extended conformation rather than an alpha-helical state on the HAP surface. Solid-state NMR approaches can thus be used to determine directly the conformation of biologically relevant peptides on HAP surfaces. A better understanding of peptide and protein conformation on biomineral surfaces may provide design principles useful for the modification of orthopedic and dental implants with coatings and biological growth factors that are designed to enhance biocompatibility with surrounding tissue.

We present a novel NMR imaging technique that allows absolute determination of the magnetic susceptibility constant, chi, of a solution. By comparing the phase difference of MR images produced with an instant (echo planar) "offset" spin-echo sequence, we obtain a direct measure of the magnetic field perturbations caused by the solution. We demonstrate this method with Gd(DTPA), Dy(DTPA), human red blood cells, and superparamagnetic iron oxide particles.