This project entails structure and dynamics studies of biological macromolecules through the use of nuclear magnetic resonance (NMR) spectroscopy. The present foci are polysaccharides, important vaccine components whose structure is poorly understood, and HIV peptides. I am using NMR to detect changes in the spectra of carbohydrates such as sucrose, cellobiose, maltose and lactose. These simple disaccharides will serve as models to provide the basis for structural studies of oligo- and polysaccharides. To date, I have found that dissolving carbohydrates in liquid crystalline media orients them in a magnetic field, and this orientation provides important and direct structural data, which previously had to be inferred. Specifically, I am measuring inter-nuclear dipolar interactions, and these interactions allow me to relate atoms to one another. One interesting outcome of the preliminary work is the suggestion that the two rings in sucrose are perpendicular. The solution structure of sucrose is still hotly debated, and these results will help answer important questions in this debate. Upon proving this methodology robust in model systems, we will study polysaccharide structure in a similar fashion. I have also verified, using C-13 nuclear relaxation measurements, that the liquid crystals and the disaccharides don't interact significantly, hence the liquid crytals are not interfering with the measurements of residual dipolar couplings. Another project is the solution structure of peptides from HIV gp41. Our goal in this work is to characterize the structure of the peptides and use them to understand the interactions which must occur to block HIV-fusion. This important region of gp41 is an excellent target for anti-HIV therapy because it is highly conserved across many strains of HIV and implied in studies as the region that is bound, by gp41 C-terminal peptides, to block viral fusion. The peptides are known to have a helical character in solution, which makes them good candidates for solution-state structural studies. To study these peptides in solution, we will need to incorporate isotopic labels (15N, and 13C) through over-expression in e. Coli. To this end Dr. Weiss has expressed DP-107 in unlabeled media and I have developed a purification scheme to provide purified DP-107. Dr. Weiss and I established a collaboration with Dr. Paul Wingfield at the Protein Expression Lab (NIAMS, NIH) to obtain labeled DP-107 for structural studies. Mass spectra of recombinant DP-107 show that it is pure and has the mass we expect. Dr. de Rosny and I are now beginning structural studies to find conditions under which a non-covalent trimer of DP-107 can be stabilized and subsequently studied by NMR.
