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Solid-state deuterium NMR spectroscopy is used to examine the dynamic behavior of 18-CD3 methyl groups in microcrystalline 6-s-cis-retinoic acid (triclinic) and 6-s-trans-retinoic acid (monoclinic) model compounds, as well as in the membrane protein bacteriorhodopsin (bR), regenerated with CD3-labeled retinal. Temperature dependent quadrupolar echo line shapes and T1 anisotropy measurements were used to characterize activation energies for 3-fold hopping motion of the methyl groups. These data provide supporting evidence that the conformation of the retinal chromophore in bR is 6-s-trans. The 6-s-cis conformer is characterized by strong eclipsing interactions between the 8-C proton and the 18-C methyl group protons; the 18-CD3 group shows an activation energy barrier for methyl 3-fold hopping of 14.5 +/- 1 kJ/mol. In contrast, the 18-CD3 group in the 6-s-trans isomer shows a considerably lower activation energy barrier of 5 +/- 1 kJ/mol. In bR, it is possible to obtain an approximate activation energy of 9 kJ/mol. This data is inconsistent with a 6-s-cis conformer but is consistent with the existence of a 6-s-trans-retinal Schiff base in bR with some interaction with the protein matrix. These results suggest that methyl rotor motions can be used to probe the van der Waals contact between a ligand and a protein binding pocket. The 6-s-trans conformer of the [16,17-(CD3)2]retinal in frozen hexane exhibits a major kinetic component with an activation energy barrier of of 14 -/+ 2 kJ/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

The application of radio frequency (RF) vacuum electronics for the betterment of the human condition began soon after the invention of the first vacuum tubes in the 1920s and has not stopped since. Today, microwave vacuum devices are powering important applications in health treatment, material and biological science, wireless communication-terrestrial and space, Earth environment remote sensing, and the promise of safe, reliable, and inexhaustible energy. This article highlights some of the exciting application frontiers of vacuum electronics.

We describe the concept of multiple-quantum cross polarization (CP) between an I = 3 2 and an I = 1 2 spin during magic-angle spinning. Experimental and theoretical results for 23 Na– 1 H pairs are presented that elucidate the transfer mechanism and the beneficial effect of adiabatic amplitude modulations of the CP field. The multiple-quantum CP approach is shown to be beneficial for improving the sensitivity of CP-MQMAS experiments and for detecting dipolar correlations.

A magic angle spinning (MAS) NMR technique for measurement of the distance between two homonuclear sites separated by as much as 0.5 nm is demonstrated. This is achieved by proton decoupling during the magnetization exchange process, greatly attenuating the influence of abundant nuclear spins, selective isotopic labeling of both sites of interest, so that the system may be approximated as a set of magnetically dilute coupled spin pairs, and MAS with matching of the rotational resonance condition. Tyrosine ethyl ester (TEE), carbon 13 labeled at both the -CH{sub 2}- of the ester moiety and at the 4{prime}-OH aromatic carbon is used as a demonstration of the technique. Calculated data is compared to experimental data.

The use of magic angle spinning nuclear magnetic resonance technique to examine local properties of four semiconductor alloys is presented. Cation distribution and charge transfer information were obtained fro the common anion systems of Cd1−xZnxTe and Hg1−xCdxTe from chemical shift, intensity, and linewidth of the nuclear magnetic resonance (NMR) signals. Preliminary results on a common cation system, CdTe1−xSex, and a dilute magnetic semiconductor, Cd1−xMnxTe, illustrate the sensitivity of NMR to local properties. In the latter case, the predominant interaction arises from transfer hyperfine interactions.

High resolution 13 C and 15 N NMR spectra have been obtained for powdered CH 3 CN. The presence of resolved dipolar structure in the 13 C spectra permits the conclusion that the symmetry axis of the 13 C shielding tensor lies along the CN bond direction.

Dipolar splittings are normally not observable because the large number of nuclear couplings usually present produces extensive broadening of the NMR lines. In this note the 13C–14N splitting in glycine is observed by using rf fields of sufficient strength to decouple the protons from the magnetically dilute nuclei. (AIP)

Proposition d'une nouvelle technique pour attenuer les bandes laterales rotationnelles sans pertes d'intensite significatives, particulierement utile pour des noyaux a grandes anisotropies des deplacements

15 N NMR spectra, obtained in the solid state by transfer of polarization from protons, are reported for (NH 4 2 SO 4 , NH 4 NO 5 , and glycine.

The voltage-dependent anion-selective channel (VDAC) is an integral membrane protein that controls transportation of metabolites across the outer mitochondrial membrane. The mechanism by which VDAC controls ion flow is important to understanding cellular metabolic processes. It remains an unresolved problem despite publication of the structure of VDAC's open conformation. According to the first gating model proposed by Marco Colombini's group, gating occurs concurrently with large structural rearrangements, and there exist multiple conformations of closed states. These closed states have not been examined by diffraction or NMR, and thus obtaining structural information on VDAC under conditions that promote closure are valuable to unraveling the gating mechanism.Accordingly we have used magic angle spinning NMR (MAS NMR) and electrophysiological measurements to study recombinant human VDAC1 in lipid bilayers under extreme pH conditions that have been shown to perturb VDAC gating and structure. Detergent-solubilzed membrane proteins are often not amenable to solution NMR studies at extreme pH conditions; thus MAS NMR is uniquely positioned to study the structure and dynamics of closed conformations of VDAC in lipid bilayers. At pH 4 and lower we observe changes in chemical shifts and peak intensities for some, but not all, residues in VDAC's N-terminus, and for residues not in the voltage-sensing domain. Changes in chemical shifts for some residues in the N-terminus are reversed when the pH is raised from low to neutral values. Electrophysiological experiments with VDAC reconstituted into a planar lipid membrane confirmed that VDAC functions properly at pH values as low as 3.0. Furthermore, low pH enhances voltage-gating, and this pH effect is fully reversible. We discuss the implications of our observations and evaluate possible gating mechanisms.

Proton decoupled 2H NMR spectra of ammonium hydrogen oxalate hemihydrate (AHOX, 5% deuterated) single crystals at room temperature have been employed to determine the electric field gradient tensors at the NH3D+, –OD (carboxyl), and HDO sites. In addition, the high resolution afforded by the narrow spectral lines (of the order of 102 Hz) and high magnetic field employed (6.8 T) made it possible to determine the chemical shift tensors of the HDO and –OD groups. The spectra show the presence of two inequivalent NH3D+ sites. At room temperature both are reorienting stepwise with rates in excess of 2×105 Hz. The motionally averaged quadrupole coupling constants for the two sites are (e2qQ/h) =5.1 kHz, η=0.53 and (e2qQ/h) =8.0 kHz, η=0.34. The quadrupole coupling and chemical shift tensor elements of the carboxyl deuterons are found to be (e2qQ/h) =129 kHz, η=0.30 and σxx=−18, σyy=−14, and σzz=6 ppm (relative to external D2O), respectively. The orientation of the tensor components can be related to the O–D bon...

A variety of novel methods in magic-angle spinning NMR is described. The effects have in common the enhancement of the influence of small anisotropies on the NMR spectrum by deliberate intervention, i.e. either by applying pulses, carefully chosen continuous r.f. fields, or by adjustment of the spinning speed. It is shown that rotational sidebands in two-dimensional spin-echo NMR are often much larger than in one-dimensional NMR, owing to the interference of the π-pulse with the rotational echo formation. It is also demonstrated that in systems containing heteronuclear spin pairs the application of a weak continuous r.f. field of carefully chosen intensity can reintroduce small heteronuclear couplings into the spectrum. This is known as rotational resonance recoupling and is due to the interference of coherent spin rotations with the normal averaging effect of the sample rotation. Related effects can occur in homonuclear spin systems when an integer multiple of the spinning speed matches the difference between isotropic chemical shifts. In this case no extra r.f. field is necessary to amplify the effect of the non-secular parts of the dipolar interaction. Greatly enhanced polarization exchange as well as strong spectral effects are demonstrated. The application of these novel methods to the measurement of small interaction tensors and thereby to the extraction of important structural information is discussed.

Gas vesicles are gas-filled organelles that allow algae, bacteria and archaea to adjust their position in the water column for optimal illumination and aeration. The spindle-shaped vesicles are typically ∼500 nm long and ∼75 nm wide, with ∼1.9 nm thick walls and their shells consist exclusively of protein, primarily the highly hydrophobic GvpA monomer (70 residues), with a permeability such that the vesicle is filled with gas of atmospheric composition. Electron microscopy has shown that the GvpA monomers are arranged in a low-pitched helix; infrared spectroscopy shows considerable beta-sheet content, in agreement with results from X-ray scattering; and atomic force microscopy shows beta-strands tilted at an angle relative to the vesicle axis that is consistent with X-ray scattering measurements on partially aligned vesicles. To gain further insight into the molecular structure and interactions that grant gas vesicles their remarkable physical properties, atomic resolution data is required. However, insolubility prevents the use of solution NMR or crystallography, and multiple scattering frustrates high-resolution electron microscopy. Here, we present the results of solid-state NMR experiments aimed at characterizing the structure of GvpA in intact, deflated gas vesicles from Anabaena flos-aquae. Fairly complete dipolar correlation spectra, indicating a largely rigid and well-ordered system, have allowed resonance assignments for ∼80% of the protein sequence. These chemical shifts provide evidence for the presence of both beta-strand and alpha-helix elements in the GvpA backbone. Furthermore, certain regions of the sequence present duplicated resonances, which suggest that the basic structural subunit of gas vesicles is an asymmetric GvpA dimer. Finally, molecular mobility and preliminary tertiary structural characteristics are also discussed.

The conversion of soluble proteins into amyloid fibrils and the structural characterization of these aggregates are outstanding problems in molecular biophysics. The amyloid-forming properties of the SH3 domain of the p85a subunit of phosphatidyl-inositol-3-kinase (PI3-SH3), an 86-residue protein, have been extensively studied in vitro by molecular biology methods. However the structure of the protein in fibril form remains unknown, and the specific structural interactions that drive its quaternary arrangement into fibrils have not been elucidated in detail. Since fibrils are insoluble and yield limited x-ray diffraction data, solid-sate NMR spectroscopy is the best-suited method with which to obtain high-resolution structural information of these systems.We present the results of solid-state NMR experiments aimed at elucidating the structure of PI3-SH3 in amyloid fibril form. The spectra of uniformly labeled PI3-SH3 present generally narrow 13C and 15N linewidths, which is characteristic of a high degree of microscopic order and structural homogeneity. Furthermore, samples grown using [2-13C] glycerol as the sole carbon source simplify the spectra and facilitate the observation of long-range internuclear correlations. Employing these alternating labeled samples and a simple data collection protocol consisting of two-dimensional homonuclear and heteronuclear correlation experiments, we have assigned ∼75% of the resonances, with the majority of unassigned residues being absent from dipolar correlation spectra due to dynamic or static disorder. Chemical shift analysis is then used to predict secondary structure elements in the PI3-SH3 sequence. The structural characteristics of PI3-SH3 fibrils revealed by solid-state NMR are described in relation to known structural and mechanistic data from previous PI3-SH3 studies.

Bacteriorhodopsin is a prototypical ion pump with a retinylidine chromophore. Ion translocation involves photo-isomerization and distortion of the chromophore, coupled with deprotonation and reprotonation of the Schiff base (SB) on opposite sides of the transport channel. Thus the SB changes its connectivity between the early and late M states, while the SB is deprotonated. Previous solid-state NMR experiments have shown that in the early M state, the SB is more strongly hydrogen-bonded than in the late M state, as indicated by the isotropic 15N chemical shifts. However, the three principle values of the chemical shift tensor are more sensitive to the environment than the isotropic average, and should yield further insight into differences between the two M states. At sufficiently low spinning frequencies, redistribution of the signal intensity from the center band to the sidebands allows calculation of the chemical shift anisotropy.View Large Image | View Hi-Res Image | Download PowerPoint SlideThe intensity of the weakest detected sideband corresponds to one site in a molecular weight of ∼500 kDa. With the signal enhancement provided by dynamic nuclear polarization, we have recorded high signal-to-noise spectra of M (see Figure) and reliably obtained the principal values of the shift tensor.