THE FOURTH DIMENSION: ACCOUNTING FOR DYNAMICS WHEN ENGINEERING ENZYMES

摘要

Despite key advances in enzyme engineering, our capacity to predict the effects of mutations on function remains nebulous. A central consideration in our incapacity to predict sequence-function relationships is the fact that proteins are dynamic, yet we rarely treat them as such. We will present methods to include dynamics in enzyme engineering. In one example, we apply computational methods to cytochrome P450 enzyme systems, to predict the trajectory of ligand binding and entry into the active-site cavity. We apply the Implicit Ligand Sampling and Adaptive Biasing Force methods to successfully predict, using a single molecular dynamics simulation, all residues known to be important for fatty acid substrate binding in cytochrome P450 BM3, thus confirming predictive accuracy. In addition, a new binding residue was identified and experimentally confirmed, and a mechanism for evolutionary protection against CO poisoning is proposed. The simulations also allow accurate docking of diverse substrates, as opposed to standardly used docking methods that are based on a single crystal structure. Finally, we successfully identify the path of substrate entry in a cytochrome P450 that has a distinct substrate preference. These new computational biology approaches show great promise to aid in identifying functional hotspots for mutation . We will also examine the distinct dynamic properties exhibited by enzymes across diverse timescales of motions. Using the β-lactamase enzyme system, we examine the effects of sequence alterations on protein dynamics on a continuum of timescales ranging from rapid side-chain fluctuations to backbone displacements. Comparison of the CPMG NMR backbone dynamics and molecular dynamics simulations of several β-lactamases reveals them to be unusually rigid, with some motions centered about the active site region. Sequence changes upon recombination in the active-site area maintained dynamics on specific timescales yet altered the dynamics on other timescales, yet catalytic function was maintained. Our results indicate that B-lactamases are highly adaptable. Furthermore, we show that enzyme engineering need not preserve all native-like dynamics in order to maintain function.