We have continued our efforts to modify and improve NMR methodologies for the assignment of resonances and the analysis of molecular structure and dynamics, and to apply these approaches to problems related to environmental health. Several recently developed methods for obtaining 1- and 2-dimensional NMR spectra have been applied to the analysis of bile acid adducts obtained subsequent to the treatment of rats with a-naphthyl isothiocyanate (ANIT) which causes bile duct necrosis. Our results indicate that the bile salt is a 5-beta, cholanic acid 3-alpha, 6-beta, 7- beta triol containing a trans CH=CH double bond between C22 and C23. The principal adduct is tauric acid. Work has continued on two structural problems, both related to the immune system. The first is a collaborative effort with Dr. Robert Handschumacher on the enzyme cyclophilin, the apparent target of the immunosuppressive drug cyclosporin A. A second collaborative study continued during the past year is aimed at determining the structure of the active site of the enzyme purine nucleoside phosphorylase (PNPase), which plays an important role in the catabolism of nucleoside drugs used to treat a variety of illnesses, and is also used in the synthesis of these drugs. The only NMR technique capable of studying complexes with enzymes this large is the transferred NOE (TRNOE), but interpretation of these experiments is difficult. We have utilized a modification of this experiment, the rotating-frame. Overhauser effect (TRROE), to reveal artifacts not easily recognized in the TRNOE results. Extensive theoretical calculations have been developed that for tile first time account for exchange kinetics. NMR methods have also been used to independently determine the rate constant for the dissociation of the enzyme-inhibitor complex, which is a critical parameter needed for the analysis of transferred NOE experiments. The results of these studies have shown that the conformation of the inhibitor bound to the enzyme is much different than that predicted on the basis of crystal structure data for the ligands themselves.
| Wallace, Bret D; Berman, Zachary; Mueller, Geoffrey A et al. (2017) APE2 Zf-GRF facilitates 3'-5' resection of DNA damage following oxidative stress. Proc Natl Acad Sci U S A 114:304-309 |
| 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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