Lab Research Focus: Structural Biology and Biomolecular NMR Spectroscopy
My laboratory is primarily interested in biomolecular NMR spectroscopy and structural biology. Recent work has centered around structure/function investigations and the characterization of motional dynamics in proteins and the development of computational methods and NMR methodologies for macromolecular structure refinement. Some of these investigations involve cross-disciplinary collaborative interactions with other research groups at UAB and elsewhere.
Protein Structural Biology: Current work is aimed at understanding the structural biology of the capsid proteins of the HIV-1 virus and other retroviruses. The wild type capsid protein of HIV-1 is particularly challenging to study by 3D/4D-NMR because of its monomer-dimer equilibrium in solution and its tendency to form oligomers. In collaboration with Dr. Prevelige of the Microbiology Department we have been studying a mutant protein that is dimerization-defective and stays as a monomer in solution, and yet is assembly active in both Gag and capsid assembly reactions. Recently we have also determined the detailed solution structure of the monomeric mutant form of the C-terminal domain of the capsid protein by 3D-NMR spectroscopy, and demonstrated that the previously known crystal structures of the dimeric forms of this protein contain dimerization-induced conformational changes in them. Ultimately, a knowledge of the structure of the capsid protein may aid in the development of novel inhibitors that interfere with the HIV-1 life cycle at the level of assembly of the mature and immature capsids. We are also interested in understanding the segmental motions (by 15N relaxation measurements) as well as the slow inter-domain motions in the capsid proteins of HIV-1 and other retroviruses. In a joint collaboration with Dr. McDonald (Pathology) we are also undertaking NMR structural studies to determine the role of calmodulin in Fas-mediated apoptosis of HIV-1 infected cells. A knowledge of the detailed three dimensional structures of calmodulin with cellular targets might lead to the development of calmodulin-antagonists that may have a therapeutic potential.
Methodological Development: Our laboratory has an active interest in developing NMR-based structure-refinement procedures. In collaboration with Dr. Istvan Sugar at the Mt. Sinai Medical Center, New York, our laboratory has developed a variable target function based intensity-restrained global optimization procedure (VARTIGO) for refining the three-dimensional structures of proteins using NOESY data. We have developed an alternative method involving Metropolis Simulated Annealing (MSA) refinement of dihedral angles against experimental NOESY intensities. Another major project in our laboratory involved the development of a Complete Relaxation and Conformational Exchange Matrix (CORCEMA) procedure for interpreting the NOESY spectra of interacting molecules such as complexes of reversibly forming ligand-receptor complexes. The CORCEMA theory is very general and is applicable over a wide range of dissociation constants for the complex (weak binding to tight binding). It incorporates explicitly all the pertinent protons of the interacting pair, and can account for the effect of motional dynamics in the complexes (e.g., hinge-bending motions in enzymes) on the NOESY. More recently, we have extended the CORCEMA theory to the saturation transfer difference (STD)-NMR experiment. This theory (CORCEMA-ST) is particularly useful in determining the bound conformations of reversibly binding low molecular weight lead compounds recognized by a target protein. Such information in turn can lead to structure-based design of new drugs. The CORCEMA-ST program developed in our laboratory for analyzing the STD-NMR data quantitatively is currently being utilized by about 62 different research groups in about 12 different countries around the world in their drug-design and developmental work. The CORCEMA and CORCEMA-ST programs may be obtained by contacting Dr. Krishna.
Biography
N. Rama Krishna is a Professor in the Department of Biochemistry and Molecular Genetics. He received his Ph.D. from the Indian Institute of Technology-Kanpur in India. He is the Director of the NMR Core Facility and holds joint appointments in the Comprehensive Cancer Center and the Departments of Chemistry and Physics. He was a Leukemia Society of America Scholar during 1982-87. His research program is supported at various times by grants from the NIH, NSF, American Heart Association, Arthritis Foundation and the Leukemia Society of America.