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. Reactivity of surface lysyl residues of proteins with a broad range of chemical agents has been proposed to be dependent on the catalytic microenvironment of the residue. We have investigated the acetylation of wild type ubiquitin, a model protein, and of the UbH68N mutant in order to evaluate the potential contribution of His-68 to the reactivity of Lys-6, which is about 4 ? distant. These studies were performed using [1-13C]acetyl salicylate or [1,1'-13C2]acetic anhydride and detecting the acetylated products by 2D HMQC NMR spectroscopy. The results demonstrate that His-68 makes a positive contribution to the rate of acetylation of Lys-6 by labeled aspirin. Additionally, a pair of transient resonances is observed after treatment of wt ubiquitin with the labeled acetic anhydride, but not upon treatment of the H68N mutant. These resonances are assigned to the acetylated His-68 residue. The loss of intensity of the acetylhistidine resonances is accompanied by an increase in intensity of the acetyl-Lys-6 peak, supporting the existence of a transacetylation process between the acetylhistidine-68 and lysine-6 residues located on the protein surface. Hence, this may be the first direct demonstration of a catalytic intermediate forming on the protein surface. Acetylation of hemoglobin by aspirin and other compounds has been of interest for the development of agents useful for the treatment of sickle cell disease. During the past year, we have used 2D NMR methods in combination with [1-13C-acetyl]salicylic acid to probe the acetylation sites of hemoglobin A and hemoglobin Tsurumai, a mutant human hemoglobin characterized by a beta-Lys-82-Gln substitution. In contrast to earlier studies by Klotz and coworkers, in which it was concluded that beta-Lys-144 is the principal target residue acetylated by aspirin, the present study confirms our previous but less conclusive demonstration that bLys-82 is the primary acetylation site of aspirin and related agents. The present studies also provide conclusive evidence that acetylation of beta-Lys-82 produces multiple resonances, probably as a consequence of additional acetylation of other sites, particularly beta-Lys-82? on the second beta chain. These results also resolve the apparent discrepancy between the targets of modification by aspirin and double headed aspirin analogs, and provide an explanation for the changes in oxygen affinity and aggregation threshold of aspirin-modified hemoglobin previously observed under in vitro conditions.
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| Gabel, Scott A; Smith, Cassandra E; Cuneo, Matthew J et al. (2014) Characterization of the redox transition of the XRCC1 N-terminal domain. Structure 22:1754-1763 |
| Gabel, Scott A; DeRose, Eugene F; London, Robert E (2013) XRCC1 interaction with the REV1 C-terminal domain suggests a role in post replication repair. DNA Repair (Amst) 12:1105-13 |
| Loeffler, Paul A; Cuneo, Matthew J; Mueller, Geoffrey A et al. (2011) Structural studies of the PARP-1 BRCT domain. BMC Struct Biol 11:37 |
| Butterfoss, Glenn L; DeRose, Eugene F; Gabel, Scott A et al. (2010) Conformational dependence of 13C shielding and coupling constants for methionine methyl groups. J Biomol NMR 48:31-47 |
| London, Robert E; Wingad, Brett D; Mueller, Geoffrey A (2008) Dependence of amino acid side chain 13C shifts on dihedral angle: application to conformational analysis. J Am Chem Soc 130:11097-105 |
| DeRose, Eugene F; Clarkson, Michael W; Gilmore, Steven A et al. (2007) Solution structure of polymerase mu's BRCT Domain reveals an element essential for its role in nonhomologous end joining. Biochemistry 46:12100-10 |
| 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 |
| Krahn, Joseph M; Jackson, Michael R; DeRose, Eugene F et al. (2007) Crystal structure of a type II dihydrofolate reductase catalytic ternary complex. Biochemistry 46:14878-88 |
| 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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