This R35 application describes our continuing and expanding program to develop and apply computationalmethods to study how protein dynamics on many timescales contributes to enzymatic catalysis and how someenzymes are crafted by evolution to make use of protein dynamics on multiple timescales. This knowledge willeventually inform approaches to design artificial enzymes a grand challenge as yet unmet. Our studies of enzymatic catalysis began years ago with the first application of Transition Path Samplingto chemical reaction in enzymes. The generation of reactive trajectory ensembles along with reactioncoordinate identification allowed us to postulate the concept of the protein promoting vibration: rapid proteindynamics at or near the active site that are directly coupled to passage over the transition state barrier toreaction. Such motions were found in multiple enzyme systems (but not all) and their importance was verifiedby experimental collaborators. More recently application of these methods to artificial enzymes that aresubjected to optimization by laboratory evolution has shown the evolutionary process introduces such motioninto a static design and we have identified protein structural changes that allows the creation and coupling ofthe dynamics. Theozymes created by de novo static structural methods have had limited success whilelaboratory evolution has allowed these proteins to develop significant catalytic power We will continue and significantly expand our program on this challenging topic through extensions of bothmethodologies employed and with application to both natural and laboratory evolved enzyme families. Inaddition to understanding how protein evolution leads to the coupling of rapid dynamics to barrier passage wewill extend our approaches to be able to map how motions far more remote in time from the passage over thechemical barrier can also be central to function. We will also develop methods that demonstrate how rapidmotions can prime the system for millisecond conformational change. Our goals for the program are tounderstand how protein dynamics ranging from sub picosecond promoting vibrations to microsecond domainmotions to millisecond conformational motion are potentially inter-related and help form enzyme function andhow such motions are orchestrated by the structure crafted by evolution. The development of such tools coupled with studies of both laboratory and naturally evolved enzymefamilies will allow the isolation of a dynamics toolbox employed by selection. We have already found how inone laboratory evolved enzyme the introduction and loss of hydrogen bonds in strategic locations results in thecreation of a promoting vibration; successful completion of the proposed program will expand suchinvestigation to the full range of protein motion timescales appropriate to catalytic turnover along with thearchitectural changes needed to create such dynamics in the protein catalyst.