Functional Dynamics and Activation Mechanisms in Enzymes

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Enzymes sample states primed to bind substrates, form Michaelis intermediates, adopt conformers facilitating chemical steps, describe all conformers with atomic resolution along the entire reaction coordinate pathway.******While many enzymes could be studied, we focus on fluoroacetate dehalogenase (FAcD) as a model enzyme namely because it is a homodimer (the most common motif among proteins) thus inviting questions regarding intra- and intermolecular allostery, which is at the heart of protein function FacD also detoxifies a fluorinated poison We will address:******1. The dynamic equilibrium of functional states. We will identify key functional states, determine the ensemble free energy landscape with a focus on interstate dynamics & solve a structure of at least one excited state (poised for substrate capture) for which there is no crystal structure. ******2. Allosteric networks & mechanisms. Methyl (TROSY) & 19F NMR will provide a network of chemical shifts across the dimer. Covariance analysis of chemical shifts as a function of inhibitor, substrate analogue, & D2O will generate an allosteric network & address the critical role of hydrogen-bonded waters as allosteric switches. ******3. Enzyme dynamics & catalysis. Dynamics will be assessed across the dimer via backbone & side chain relaxation experiments. We will study the connection between catalytic efficiency & molecular dynamics. ******4. Substrate inhibition. We will study how an allosteric substrate pocket in the dimer contributes to inhibition at high concentrations & efficient catalysis at low concentrations. ******5. 19F NMR assignments crystallography. We will improve the use of DFT & local MD simulations as an alternative to mutagenesis for 19F NMR assignments.******6. Protein folding of a dimer. Both denaturant & pressure will be used to study the complex folding process of this dimer with atomic resolution. ******7. Protein mutagenesis & evolution. High numbers of variants will be made & studied via single cell (FACS) methods. Allosteric networks will be tested & the enzyme will be evolved toward greater catalytic efficiency or substrate promiscuity. ******Results will greatly advance understanding of the role of structure & dynamics in catalysis from the perspective of an ensemble, while addressing several paradigm shifting questions regarding allostery & water networks in a prototypical dimer.