Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates.

Can non-mechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation.

Atomic force microscopy (AFM) experiments have provided intriguing insights into the mechanical unfolding of proteins such as titin I27 from muscle, but will the same be possible for proteins that are not physiologically required to resist force? We report the results of AFM experiments on the forced unfolding of barnase in a chimeric construct with I27. Both modules are independently folded and stable in this construct and have the same thermodynamic and kinetic properties as the isolated proteins. I27 can be identified in the AFM traces based on its previous characterization, and distinct, irregular low-force peaks are observed for barnase. Molecular dynamics simulations of barnase unfolding also show that it unfolds at lower forces than proteins with mechanical function. The unfolding pathway involves the unraveling of the protein from the termini, with much more native-like secondary and tertiary structure being retained in the transition state than is observed in simulations of thermal unfolding or experimentally, using chemical denaturant. Our results suggest that proteins that are not selected for tensile strength may not resist force in the same way as those that are, and that proteins with similar unfolding rates in solution need not have comparable unfolding properties under force.