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 or pharmacological concern. The formation of protein adducts by environmental agents involves complex chemical/biochemical/structural interactions which are often incompletely understood. This project has involved studies on adduct formation by a model protein, ubiquitin, as well as a number of other applications relevant to environmental or pharmacological problems. NMR studies of the interaction of [acetyl-1- 13C] aspirin with the model protein ubiquitin have demonstrated that: 1. Relative rates of modification for K6, K48, K63, K11, K33, and M1 were found to be 1.0: 0.59: 0.43: 0.26: 0.23: 0.03. 2. These rates indicate that reactivity of the amino groups of ubiquitin is determined primarily, although not exclusively, by residue accessibility. Recent studies of the acetylation of the H68N ubiquitin mutant by labeled acetic anhydride have indicated that a transient acetyl-histidine adduct can be observed. Evidence for the importance of H68 in explaining the high acetylation rate of K6 has also been obtained from studies of this mutant. Studies of reductively methylated ubiquitin indicate the presence of long range interactions and exhibit interesting pH dependent effects. Analysis of the structural perturbation of the ubiquitin R54L mutation by NMR indicates that the structural perturbations are localized to the region near the substitution: in particular the Glu51-Asp-Gly-Arg54 reverse turn and the Ile23 and Glu24 on helix 1. Alternatively, the more conservative R11K substitution has longer range effects, presumably due to alteration of the R11 - E34 salt bridge interaction. Studies of human hemoglobin modifed with labeled aspirin or methyl acetyl phosphate have demonstrated that: 1. In contrast with previous evaluations, aspirin acetylation targets primarily beta-Lys82 in the 2,3-diphosphoglycerate binding pocket; 2. Acetylation by methyl acetyl phosphate gives a residue profile which is fairly similar that observed for acetylation by aspirin. Although it has been suggested that the pharmacokinetics of aspirin make it unsuitable for modification of any but the most reactive targets, the very long lifetime of hemoglobin in erythrocytes makes cumulative acetylation a feasible clinical objective. - aspirin; ubiquitin; hemoglobin; sickle cell hemoglobin; protein adducts; nuclear magnetic resonance
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| DellaVecchia, Matthew J; Merritt, W Keither; Peng, Ye et al. (2007) NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB-domain 4 heterodimer formation in solution. J Mol Biol 373:282-95 |
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| London, Robert E; Gabel, Scott A (2006) Photoactivated h/d exchange in tyrosine: involvement of a radical anion intermediate. J Am Chem Soc 128:2268-75 |
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