Protein dynamics from femtoseconds to milliseconds as crafted by natural and laboratory evolution: towards enzyme design

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This R35 application describes our continuing and expanding program to develop and apply computational methods to study how protein dynamics on many timescales contributes to enzymatic catalysis, and how some enzymes are crafted by evolution to make use of protein dynamics on multiple timescales. This knowledge will eventually 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 Sampling to chemical reaction in enzymes. The generation of reactive trajectory ensembles along with reaction coordinate identification allowed us to postulate the concept of the protein promoting vibration: rapid protein dynamics at or near the active site that are directly coupled to passage over the transition state barrier to reaction. Such motions were found in multiple enzyme systems (but not all,) and their importance was verified by experimental collaborators. More recently, application of these methods to artificial enzymes that are subjected to optimization by laboratory evolution has shown the evolutionary process introduces such motion into a static design and we have identified protein structural changes that allows the creation and coupling of the dynamics. “Theozymes” created by de novo static structural methods have had limited success, while laboratory 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 both methodologies employed and with application to both natural and laboratory evolved enzyme families. In addition to understanding how protein evolution leads to the coupling of rapid dynamics to barrier passage, we will extend our approaches to be able to map how motions far more remote in time from the passage over the chemical barrier can also be central to function. We will also develop methods that demonstrate how rapid motions can prime the system for millisecond conformational change. Our goals for the program are to understand how protein dynamics ranging from sub picosecond promoting vibrations to microsecond domain motions to millisecond conformational motion are potentially inter-related and help form enzyme function, and how such motions are orchestrated by the structure crafted by evolution. The development of such tools coupled with studies of both laboratory and naturally evolved enzyme families will allow the isolation of a “dynamics toolbox” employed by selection. We have already found how in one laboratory evolved enzyme the introduction and loss of hydrogen bonds in strategic locations results in the creation of a promoting vibration; successful completion of the proposed program will expand such investigation to the full range of protein motion timescales appropriate to catalytic turnover, along with the architectural changes needed to create such dynamics in the protein catalyst.