Exploration of the activation pathway of Deltaalpha-Chymotrypsin with molecular dynamics simulations and correlation with kinetic experiments.

Correlating the experimentally observed kinetics of protein conformational changes with theoretical predictions is a formidable and challenging task, due to the multitude of degrees of freedom (>5,000) in a protein and the huge gap between the timescale of the kinetic event of interest (ms) and the typical timescale of computer simulations (ns). In this study we show that using the targeted molecular dynamics (TMD) method it is possible to simulate conformational changes of the ms time range and to correlate multiple simulations of single pathways with ensemble experiments on both the structural and energetic basis. As a model system we chose to study the conformational change of rat-Deltaalpha-chymotrypsin from its inactive to its active conformation. This activation process has been analyzed previously by experimental and theoretical methods, i.e. fluorescence stopped-flow spectroscopy (FSF), molecular dynamics (MD) and TMD. Inspired by the results of these studies on the wild type (WT) enzyme, several mutants were constructed to alter the conformational pathway and studied by FSF measurements. In the present work WT and mutant N18G were subjected to multiple MD and subsequent TMD simulations. We report the existence of two main activation pathways, a feature of chymotrypsin activation that has been hitherto unknown. A method to correlate the energetics of the different pathways calculated by TMD and the kinetic parameters observed by experimental methods such as FSF is presented. Our work is relevant for experimental single molecule studies of enzymes in general.