NMR STUDIES OF BIOMOLECULAR STRUCTURE, FUNCTION, AND DYNAMICS

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Summary of Work: The overall aim of this project involves the use of nuclear magnetic resonance (NMR) spectroscopy to characterize biological macromolecules and their interaction with compounds of environmental concern. The formation of protein adducts by environmental agents involves complex chemical/biochemical/structural interactions which are, at best, incompletely understood. Work to date has involved several agents of environmental and pharmacological interest, including bromoacetate, a carboxymethylating agent, and aspirin. Two dimensional proton-carbon HMQC experiments allow resolution of the adduct resonances formed from reaction with individual residues. Several approaches to the assignment of these resonances have been evaluated. These studies have involved two target proteins: ubiquitin - a small, stable protein lacking an active site, and, more recently, hemoglobin. Although the primary physiological target of acetylation by aspirin is believed to be prostaglandin synthase, aspirin is known to acetylate many other proteins, e.g. albumin and hemoglobin, and such interactions can result in a broad range of pharmacological and toxicological effects. The study of hemoglobin was recently initiated based on discussions with Dr. Richard Labotka who has been investigating various agents capable of interfering with the aggregation of sickle hemoglobin. Aspirin was one of the earliest agents studied for this purpose, and is known to modify residues in the DPG binding pocket. Dr. Labotka has recently developed several improved agents for this purpose, and we are studying hemoglobin modifcation by these agents as a collaborative project. Relative to aspirin and other agents which have been evaluated for this purpose, Dr. Labotka's compounds are predicted to have improved transport properties and should be less perturbing to normal cellular metabolism. A collaborative research project with Dr. Linda Luck (Clarkson University) on the E. coli periplasmic glucose-galactose receptor has continued. A manuscript based on the dynamic frequency shift behavior observed for [1- 13C,1-2H]glucose complexed with the receptor was recently completed, and further studies of fluorotryptophan labeled receptor are in progress.