Determination of the chemical shielding tensor orientation from two or one of the three conventional rotations of a single crystal.

Chemical shift anisotropy (CSA) is an immensely useful interaction to study the structure, dynamics, and function of a wide variety of chemical and biological molecules. Traditionally the only unambiguous way to determine both the principal values and the orientation of the principal axes of the CSA tensor has been to follow the chemical shift frequency changes as a crystal of known structure is rotated relative to the direction of the external magnetic field. This classic method employs rotations about three mutually orthogonal axes of a single crystal. It is shown here that just two, or one, of the above rotations suffice to determine the CSA tensor orientation by borrowing, the easy to obtain, principal values of CSA from an independent source. Methods for using two rotation patterns or even a single rotation pattern are described and illustrated with known chemical shielding tensors.

Transmembrane domain of M2 protein from influenza A virus studied by solid-state (15)N polarization inversion spin exchange at magic angle NMR.

The M2 protein from the influenza A virus forms a proton channel in the virion that is essential for infection. This tetrameric protein appears to form a four-helix bundle spanning the viral membrane. Here the solid-state NMR method, 2D polarization inversion spin exchange at magic angle (PISEMA), has been used to obtain multiple constraints from specifically amino acid-labeled samples. The improvement of spectral resolution from 2D PISEMA over 1D methods and 2D separated local field methods is substantial. The reliability of the method is validated by comparison of anisotropic chemical shift and heteronuclear dipolar interactions from single site labeled samples. The quantitative interpretation of the high-resolution constraints confirms the helix tilt to be within the range of previous experimental determinations (32 degrees -38 degrees ). The binding of the channel inhibitor, amantadine, results in no change in the backbone structure at position Val(27,28), which is thought to be a potential binding site for the inhibitor.

Internal packing of helical membrane proteins.

Helix packing is important in the folding, stability, and association of membrane proteins. Packing analysis of the helical portions of 7 integral membrane proteins and 37 soluble proteins show that the helices in membrane proteins have higher packing values (0.431) than in soluble proteins (0.405). The highest packing values in integral membrane proteins originate from small hydrophobic (G and A) and small hydroxyl-containing (S and T) amino acids, whereas in soluble proteins large hydrophobic and aromatic residues have the highest packing values. The highest packing values for membrane proteins are found in the transmembrane helix-helix interfaces. Glycine and alanine have the highest occurrence among the buried amino acids in membrane proteins, whereas leucine and alanine are the most common buried residue in soluble proteins. These observations are consistent with a shorter axial separation between helices in membrane proteins. The tight helix packing revealed in this analysis contributes to membrane protein stability and likely compensates for the lack of the hydrophobic effect as a driving force for helix-helix association in membranes.

Rotational resonance NMR of 13C2-labelled retinal: quantitative internuclear distance determination.

Rotational resonance phenomena are investigated in the solid-state magic-angle spinning NMR of all-E-[11,20-13C2]-retinal at a magnetic field of 4.7 T. We find good agreement between experiments and numerical simulations for the rotational resonance spectral peakshapes and for the rotor-driven magnetization exchange. The internuclear distance between the 13C-labelled C11 and C20 sites is determined to be 0.301 +/- 0.008 nm (from rotational resonance spectra) and 0.300 +/- 0.010 nm (from rotor-driven magnetization exchange), in agreement with the X-ray crystallographic distance of 0.296 nm. We show rotational resonance spectra which display perturbations from intermolecular homonuclear spin-spin interactions.

Conformational transitions of the phosphodiester backbone in native DNA: two-dimensional magic-angle-spinning 31P-NMR of DNA fibers.

Solid-state 31P-NMR is used to investigate the orientation of the phosphodiester backbone in NaDNA-, LiDNA-, MgDNA-, and NaDNA-netropsin fibers. The results for A- and B-DNA agree with previous interpretations. We verify that the binding of netropsin to NaDNA stabilizes the B form, and find that in NaDNA, most of the phosphate groups adopt a conformation typical of the A form, although there are minor components with phosphate orientations close to the B form. For LiDNA and MgDNA samples, on the other hand, we find phosphate conformations that are in variance with previous models. These samples display x-ray diffraction patterns that correspond to C-DNA. However, we find two distinct phosphate orientations in these samples, one resembling that in B-DNA, and one displaying a twist of the PO4 groups about the O3-P-O4 bisectors. The latter conformation is not in accordance with previous models of C-DNA structure.

Interaction of cyclosporin A with dipalmitoylphosphatidylcholine.

Cyclosporin A, a hydrophobic cyclic peptide, is a potent immunosuppressant. In an attempt to determine the localization of cyclosporin A in phospholipid membranes, the effect of cyclosporin A on dipalmitoylphosphatidylcholine (DPPC) has been investigated using deuterium nuclear magnetic resonance (2H-NMR) spectroscopy and differential scanning calorimetry (DSC). Cyclosporin A was dispersed within acyl chain per-deuterated DPPC at a concentration of 6 mole percent, hydrated with buffer, and the spectra obtained over a range of temperatures were compared with that of pure DPPC. The changes caused by cyclosporin A were assessed by the first moment (M1) and order parameters calculated from the spectra. The presence of cyclosporin A decreases the magnitude of M1 at temperatures below the gel to liquid-crystalline phase transition temperature but increases M1 at temperatures above the transition. In addition, the change in M1 at the transition temperature was also less abrupt when cyclosporin A was present. For bilayers in the liquid-crystalline state, cyclosporin A causes an increase in the order parameters along the acyl chains which suggests that cyclosporin A is located along the acyl chains of the phospholipid. For DSC, cyclosporin A was dispersed in non-deuterated DPPC at different peptide to phospholipid mole ratios. The endothermic peaks associated with the gel to liquid-crystalline phase transition and pretransition were recorded and compared with similar mole ratios of cholesterol to lipid. At 30 mole percent cyclosporin A, small decreases in the main transition temperature and associated enthalpy were observed, whereas at 30 mole percent cholesterol, the main transition is barely distinguishable from the baseline. The pretransition was not observed with the addition of 11 mole percent of either cyclosporin A or cholesterol. The results of the thermal analysis indicate that although cyclosporin A and cholesterol appear to be both located along the acyl chains of the phospholipids, they have dramatically different interactions with the membrane lipids.

Pyrrolidine ring conformations in prolyl peptides from 13C spin-lattice relaxation times.

A method was developed in the framework of a bistable jump model to obtain the pyrrolidine ring conformations in proline peptides from 13C spin-lattice relaxation times. Equations are presented expressing the ring torsions in terms of the 13C spin-lattice relaxation times of the ring carbons. This method was applied to 26 pyrrolidine ring systems and acceptable conformations were obtained.