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, & acquire states enabling product release, while minimizing the reverse process. Catalysis is enabled by a dynamic equilibrium of functional states, referred to as the ensemble. NMR goes beyond crystallography to capture high-resolution structures of ground & excited states in addition to both fast (local) & slow (cooperative) dynamics enabling catalysis. The ensemble perspective is both profoundly important to advancing our understanding of protein function & profoundly challenging, since we seek to 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 & least understood.  FacD also detoxifies a fluorinated poison & represents an interesting model to pursue mutagenesis, functional evolution, & bioremediation.  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.