Christian G. Canlas

Solid state NMR measurements of conformation and conformational distributions in the membrane-bound HIV-1 fusion peptide.

The solid state NMR lineshape of a protein backbone carbonyl nucleus is a general diagnostic of the local conformational distribution in the vicinity of that nucleus. In addition, measurements of carbonyl chemical shifts and 2D exchange spectra provide information about the most probable conformation in the distribution. These types of solid state NMR methodologies have been applied to structural studies of the membrane-bound HIV-1 fusion peptide. This peptide is derived from a domain of the HIV-1 gp41 envelope protein, which is critical for viral-host cell-membrane fusion. Even in the absence of the rest of the envelope protein, the fusion peptide will fuse liposomes or erythrocytes. The solid state NMR measurements demonstrate that the center of the membrane-bound HIV-1 fusion peptide is structured, while the C-terminus is highly disordered. The structural distribution at the peptide center is lipid-dependent, with the greatest degree of structural homogeneity in a lipid environment whose composition reflects that of the target T cells. When bound to the lipid mixture, the peptide center is predominately beta sheet. The beta-sheet structure may be diagnostic of peptide oligomerization, which is thought to be a requirement for membrane fusion activity. Although the peptide partially disrupts bilayer orientational ordering in stacked glass-plate samples, 2H NMR demonstrates that the bilayers remain intact in the presence of the fusion peptide and are not micellized. The retention of the bilayer phase may relate to the biological requirement that the virus should fuse with, but not destroy, the target host cell membrane.

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.

Nuclear magnetic resonance evidence for retention of a lamellar membrane phase with curvature in the presence of large quantities of the HIV fusion peptide

The HIV fusion peptide (HFP) is a biologically relevant model system to understand virus/host cell fusion. (2)H and (31)P NMR spectroscopies were applied to probe the structure and motion of membranes with bound HFP and with a lipid headgroup and cholesterol composition comparable to that of membranes of host cells of HIV. The lamellar phase was retained for a variety of highly fusogenic HFP constructs as well as a non-fusogenic HFP construct and for the influenza virus fusion peptide. The lamellar phase is therefore a reasonable structure for modeling the location of HFP in lipid/cholesterol dispersions. Relative to no HFP, membrane dispersions with HFP had faster (31)P transverse relaxation and faster transverse relaxation of acyl chain (2)H nuclei closest to the lipid headgroups. Relative to no HFP, mechanically aligned membrane samples with HFP had broader (31)P signals with a larger fraction of unoriented membrane. The relaxation and aligned sample data are consistent with bilayer curvature induced by the HFP which may be related to its fusion catalytic function. In some contrast to the subtle effects of HFP on a host-cell-like membrane composition, an isotropic phase was observed in dispersions rich in phosphatidylethanolamine lipids and with bound HFP.

Investigation of longitudinal 31P relaxation in metal selenophosphate compounds.

Molten salt syntheses yield a rich variety of metal selenophosphate compounds which have a wide range of 31P T(1) longitudinal relaxation times (20-3000 s). There is a qualitative positive correlation between squared dipolar couplings and 1/T(1), suggesting that these interactions contribute to relaxation. However, two of the compounds, K(2)CdP(2)Se(6) and Rb(2)CdP(2)Se(6), have T(1) which are significantly shorter than what is expected from dipolar couplings. The ESR spectra of these compounds show the presence of unpaired electrons which may accelerate the rate of 31P relaxation. The importance of relaxation in application of (31)P NMR to these systems is demonstrated in analysis of the mixture of crystalline products formed in a Ag(4)P(2)Se(6) synthesis. At short relaxation delays, the NMR intensities are non-quantitative and overestimate the concentration of an Ag(7)PSe(6) impurity.