Research Summary
The Alpha/Beta Barrels:
The eight stranded a/ b barrel protein fold is the largest family of protein structures (representing at least 10% of all known protein structures) and has the widest range of enzyme functions. My research focuses on elucidating the structural basis for folding specificity and thermodynamic stability in this class of enzymes and the future application of those principles in protein design. Specifically, I use random PCR mutagenesis to create a library of genetic variants for a variety of enzymes including beta-galactosidase. These variants are placed in an expression host that is a beta-galactosidase negative yet positive for the lactose transport genes. A temperature selection is applied. Using chromogenic substrates for screening, or nutrient restriction for selection, variants that display the selected phenotype (activity or stability at higher or lower temperature) can be selected. These variants are subjected to future rounds of mutagenesis and selection until variants with dramatically different enzymatic properties are produced. Using multiple successes from these “Directed Evolution” experiments, coupled with careful analysis of structure, patterns of mutations begin to emerge. These patterns help us posit generalized, fold-specific, molecular mechanisms of protein structural adaptation to temperature. These principles are then tested using site directed mutagenesis and rational design.
Other Folds:
The above methods are applied to enzymes of other protein fold families to determine structural adaptation strategies to temperature that are specific to those folds.
Entropic Stabilization in Thermostable Enzymes:
Using ab initio Monte Carlo simulations to estimate conformational space available to the unfolded state and that disallowed due to steric exclusion of side chain and main chain atoms, general classes of substitutions and a new model of protein thermostabilization through entropy is proposed.
