This project utilizes state-of-the-art NMR spectroscopy to study problems that are of continuing interest to the NIEHS, the Laboratory of Structural Biology, and the NMR research group. The primary emphasis involves applications in three areas: 1) Understanding how the structural and dynamic behavior of DNA polymerases relates to the fidelity of nucleotide incorporation, 2) studies of ligand-macromolecule interactions, and 3) development and evaluation of new methodologies for the structural and dynamic characterization of proteins and other biological macromolecules in support of the research goals. Progress during the past year is summarized below: Project 1. During the past year, we used NMR methods to determine the solution structure of the theta subunit of E. coli DNA polymerase III. This subunit, which forms part of the polymerase core, interacts with the proofreading exonuclease subunit epsilon. This work was prompted by our recent determination of the structure of a theta homolog present in phage P1, called """"""""HOT"""""""". Despite a high level of sequence identity between HOT and theta, our structure of HOT differs substantially from the structure of theta previously reported by another group. This difference suggested the possibility of an error in the previous determination of the theta structure, although in general, sequence similarity does not guarantee structural homology. In contrast with HOT, the 1H-15N HSQC spectrum of theta is poorly resolved, suggesting either aggregation and/or conformational exchange behavior in solution. A solution to this problem was suggested by a homology model of theta based on the structure of HOT, according to which, several hydrophobic residues of theta extend into solution, potentially leading to solubility and aggregation problems. Based on this model, the solvent was varied from 100 % water to 60:40 water:methanol. The quality of the HSQC spectrum improved significantly under the latter conditions, allowing the structure determination to proceed. As anticipated, it was determined that the poor spectral quality had resulted in a significant error in the previous structural determination. It is anticipated that the availability of this structural information will provide insight into the different mutation profile observed in E. coli when plasmid-encoded HOT is introduced as a substitute for theta. Project 2. Studies of ligand-macromolecule interactions have continued to focus on the Type II dihydrdofolate reductase, R67 DHFR, a plasmid encoded enzyme which confers resistance to anti-folate drugs on the bacteria containing the plasmid. During the past year, we have characterized the binding of numerous analogs, inhibitors and fragments of NADPH and/or folate by isothermal titration calorimetry, nuclear magentic resonance, and X-ray crystallography. As anticipated, reducing the length of the molecule generally resulted in reduced affinity. A buffer dependence for binding of folate was also observed, which correlates with perturbation of the N3 pKa, such that binding of a neutral pteridine ring is preferred. In addition to these calorimetric studies, we have obtained preliminary data on the structure of the ternary R67 DHFR?NADP?dibromofolate complex. These studies suggest that the type II DHFR is able to recognize the substrate and cofactor by a parallel mode of binding that involves interaction of Ile68 carbonyl and amide groups with the carboxamide group of NADP and with the N3-O4 amide group of the pteridine ring system. Crystallography and inter-ligand Overhauser effect studies show that the two ring systems adopt a relative endo geometry, characterized by tilted aromatic rings which approach each other most closely at nicotinamide C4-pteridine C6, corresponding to the reactive positions on the corresponding substrates. This binding mode differs from that observed in Type I DHFR, but is similar to that recently observed in pteridine reductase. This study provides a basis for understanding trimethoprim resistance conferred by the enzyme and for the development of Type II DHFR-targeted inhibitors. Project 3. The development of NMR methods for the analysis of protein structure has been intimately linked with isotopic labeling strategies. Although uniform labeling with 13C, 15N and occasionally 2H is extremely useful for smaller proteins, the structural analysis of proteins with MW > 25,000 has often required specific labeling approaches. We have recently investigated the photochemical H/D exchange reaction in tyrosine in order to obtain useful labeling patterns for structural NMR studies.
| 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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