CAREER: NMR Studies of Enzyme Structure and Dynamics

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Foster, Mark P.MCB-0092962The objective of this CAREER project is to reveal how molecular motions affect enzyme function. The limited knowledge of structure/motion/function relationships severely limits our ability to predict molecular behavior. This knowledge is critical both for understanding catalytic mechanisms and for the design of modified enzymes that catalyze novel chemistries or specific high-affinity ligands. In this project, functionally important molecular motions in bacteriophage l-Integrase are characterized with an integrated approach involving nuclear magnetic resonance (NMR), pre-steady state kinetics, thermodynamics, computation and site-directed mutagenesis. Biochemical and biophysical evidence suggests that large amplitude concerted loop motions are essential to the function of this enzyme, which catalyzes site-specific DNA recombination via the formation of a Holliday junction intermediate. To study these motion/function relationships the project includes: (1) Comparison of the equilibrium structures, dynamics and thermodynamics of the free and DNA-bound enzyme in order to identify the nature of the conformational changes required for catalysis. (2) Measurement of the kinetics and thermodynamics of DNA binding and cleavage with NMR, mass spectrometry, fluorescence and calorimetry. (3) Obtaining a physical description of relevant internal motions from molecular dynamics simulations. (4) Probing the dependence of function on internal motions by use of site-directed mutagenesis and alternate DNA substrates.The educational component of this CAREER award involves development of descriptive tools to enable scientists and educators to effectively communicate to students the dynamic nature of macromolecules. Currently most didactic treatments of molecular structure/function are limited to static pictures that provide little insight into the nature of the motional processes that interconvert functional substates.