Enzyme function relies on the ability of the polypeptide backbone to maintain a proper three-dimensional fold and yet retain enough flexibility to bind substrates, catalyze the requisite reaction and release products. Understanding the physical principles that balance flexibility and rigidity is essential for elucidation of the function of enzymes, de novo design of protein biocatalysts, development of new protein scaffolds and optimization of drug/protein interactions. The overall goal of the research proposed here is to employ a powerful combination of biochemical techniques and solution NMR spectroscopy, including nuclear spin-relaxation measurements, to characterize the interrelationship between enzyme function and protein conformational fluctuations. In particular, the proposed work is directed at study of the protein VanX, a D-,D-amino acid dipeptidase whose action is essential in the resistance of Gram-positive bacteria to the antibiotic vancomycin. Vancomycin has been an effective antibiotic, however, the emergence of resistant bacterial strains is cause for concern. VanX, like many proteases, undergoes a slow conformational isomerization upon interaction with tight-binding inhibitors. This proposal specifically aims to characterize the physico-chemical properties of VanX that facilitate this structural transformation. NMR spin-relaxation measurements will be used to characterize rates and energy barriers of VanX motional dynamics. Combination of these studies with biochemical interrogation of enzyme kinetics and ligand binding will be used establish a correlation between motional and enzymatic dynamics.
| Kovrigin, Evgenii L; Kempf, James G; Grey, Michael J et al. (2006) Faithful estimation of dynamics parameters from CPMG relaxation dispersion measurements. J Magn Reson 180:93-104 |
| Kempf, James G; Loria, J Patrick (2004) Measurement of intermediate exchange phenomena. Methods Mol Biol 278:185-231 |
| Kempf, James G; Jung, Ju-yeon; Sampson, Nicole S et al. (2003) Off-resonance TROSY (R1 rho - R1) for quantitation of fast exchange processes in large proteins. J Am Chem Soc 125:12064-5 |
| Kempf, James G; Loria, J Patrick (2003) Protein dynamics from solution NMR: theory and applications. Cell Biochem Biophys 37:187-211 |
