Motions are key to the action of many proteins. Dynamics are poorly understood, especially, in the micro- second and slower regimes that often limit turnover. They lie beyond most experimental and computational methods. Our long term objective is a fundamental understanding of how conformational dynamics underpin mechanism. This will be achieved by characterizing structurally and kinetically the motions of individual amino acids and their relation to function in the turnover cycle of an induced-fit enzyme. Recently developed methods of NMR relaxation dispersion analysis will be applied to a model enzyme to determine conformational exchange rates. These will be integrated with high resolution crystal structures and NMR residual dipolar coupling characterization of faster dynamics. Arginine kinase presents examples of domain and loop movements that are substrate-induced, as well as inherent concerted motions. Both the NMR spectra and x-ray diffraction of arginine kinase are of unusually high quality for a representative enzyme of this size (42 kDa). This presents a unique opportunity to elucidate the structure-dynamics- function correlates of motions representative of those that are functionally important for many proteins. The emphasis will be on mapping the backbone conformational exchange rates for each residue, particularly in the micro-/milli-second regime. NMR relaxation exchange rates will be measured during enzyme turnover at various equilibrium substrate/product concentrations to determine which motions are associated with binding, dissociation or reaction steps. Crystal structures will be determined for the stable enzyme complexes within the random sequential bi-bi mechanism, and NMR relaxation titration will be used to determine the exchange rates for conformational changes associated with binding or dissociation. Thus, the structural changes and time constants of motion will be dissected for each amino acid through the steps of the turnover cycle. The functional role of the motions will be established through joint analysis of kinetics and dynamics following temperature, mutational and other perturbations of the enzyme. Health Relevance: A fundamental understanding of molecular motion is essential to understanding how human proteins achieve their normal function and the mechanisms through which disease may result from genetic mutations.
| Davulcu, Omar; Peng, Yu; Brüschweiler, Rafael et al. (2017) Elevated ?s-ms timescale backbone dynamics in the transition state analog form of arginine kinase. J Struct Biol 200:258-266 |
| Peng, Yu; Hansen, Alexandar L; Bruschweiler-Li, Lei et al. (2017) The Michaelis Complex of Arginine Kinase Samples the Transition State at a Frequency That Matches the Catalytic Rate. J Am Chem Soc 139:4846-4853 |
| Gu, Yina; Hansen, Alexandar L; Peng, Yu et al. (2016) Rapid Determination of Fast Protein Dynamics from NMR Chemical Exchange Saturation Transfer Data. Angew Chem Int Ed Engl 55:3117-9 |
| Godsey, Michael H; Davulcu, Omar; Nix, Jay C et al. (2016) The Sampling of Conformational Dynamics in Ambient-Temperature Crystal Structures of Arginine Kinase. Structure 24:1658-1667 |
| Hansen, Alexandar L; Brüschweiler, Rafael (2016) Absolute Minimal Sampling in High-Dimensional NMR Spectroscopy. Angew Chem Int Ed Engl 55:14169-14172 |
| Chapman, Brynmor K; Davulcu, Omar; Skalicky, Jack J et al. (2015) Parsimony in Protein Conformational Change. Structure 23:1190-8 |
| Summerton, Jean C; Martin, Gregory M; Evanseck, Jeffrey D et al. (2014) Common hydrogen bond interactions in diverse phosphoryl transfer active sites. PLoS One 9:e108310 |
| Davulcu, Omar; Niu, Xiaogang; Brüschweiler-Li, Lei et al. (2014) Backbone resonance assignments of the 42 kDa enzyme arginine kinase in the transition state analogue form. Biomol NMR Assign 8:335-8 |
| Summerton, Jean C; Evanseck, Jeffrey D; Chapman, Michael S (2012) Hyperconjugation-mediated solvent effects in phosphoanhydride bonds. J Phys Chem A 116:10209-17 |
| Clark, Shawn A; Davulcu, Omar; Chapman, Michael S (2012) Crystal structures of arginine kinase in complex with ADP, nitrate, and various phosphagen analogs. Biochem Biophys Res Commun 427:212-7 |
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