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.
Proteins. Our laboratory has been active in determining the three-dimensional structures of long-chain neurotoxic proteins derived from Centruroides sculpturatus Ewing (range southwestern USA). These studies can be linked to diverse toxicities and effects on the sodium channels exhibited by these interesting proteins. More recently, we have determined the structures of two separate Old-World-like neurotoxins from the venom of this New World scorpion, as examples of evolutionary carryover. We have also determined the first three-dimensional structure of a beta-toxin (CsE-I). The dynamical behaviour of these proteins can differ enormously from each other, despite good sequence homology. Efforts are underway to express these proteins with uniform isotopic labeling by recombinant methods. Our long term goals are to study the interactions of these proteins with extracellular receptor domains on the sodium channel. We are also investigating the solution structure of interferon-tau (IFN-tau), a type-I interferon that is unusual in that it is not toxic even in high doses, unlike other interferons. Interferon-tau exhibits antiviral and antiproliferative properties. We have successfully cloned and expressed the uniformly labeled protein. We plan to ultimately elucidate the interactions between IFN-tau with type-I IFN receptor complex. In collaboration with Dr. Peter Prevelige at UAB we are also investigating the structure-function relationships of the scaffolding protein (303 residues) from bacteriophage P22, a dsDNA virus. We have already determined the detailed three dimensional structure of the coat protein-binding domain of this scaffolding protein by 3D-NMR. These studies are now being extended to larger fragments containing dimerization and coat protein-binding domains.
Methodological Development. Our laboratory has an active interest in 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. The CORCEMA program is currently being used in the quantitative analysis of transferred NOESY spectra of ligand/protein complexes at several laboratories in USA and abroad. A copy of the CORCEMA program may be obtained by contacting Dr. Krishna. More recently, we have extended the CORCEMA theory to the saturation transfer difference (STD)-NMR experiment.
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.
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