Instrumentation and methods development for millisecond time-resolved studies of protein dynamics using quenching crystallography

Cornell University, Ithaca NY

Investigators

Abstract

An award is made to Cornell University to develop new technology and methods for time-resolved study of proteins in action. Proteins are dynamic machines that interact with other molecules to perform myriad functions required for life. Three dimensional structures of proteins determined using X-ray crystallography reveal their atomic construction, provide insight into how they function and malfunction, and provide the basis for understanding the molecular mechanisms of protein function. Even more could be learned if these static structures could be complemented by “molecular movies” of proteins in action, for example, as they catalyze chemical reactions to generate new molecules. The technology to be developed here promises to provide an effective and efficient approach to making these “movies” with millisecond time resolution, allowing time-resolved crystallography to be applied to far more biomolecular targets by more researchers at more diverse institutions. This development work will engage a diverse science and engineering team, with efforts made to recruit participants who will benefit most from the hands-on and interdisciplinary training provided. If this work is successful, there is a clear path for commercial development to maximize the technology’s impact. These research efforts are complemented by longstanding efforts in undergraduate education and teacher training. Powerful methods using free-electron laser X-ray sources have been developed for time-resolved study of biomolecular reactions initiated by light or by chemical mixing, but their application is resource intensive and not well suited to broad scientific and biotechnological use. An alternative approach has been demonstrated in which reactions initiated in crystals are quenched within tens of milliseconds by ultra-rapid cooling, and X-ray data collected from these crystals via standard mail-in cryocrystallography. This approach separates the timescale of the reaction from that of X-ray data collection, yields more useful data per crystal, and minimizes the number of crystals required for structure determination at each time point. Here, instrumentation and methods for reaction initiation and rapid cooling will be developed to decrease achievable time resolution toward 1 ms, and key parameters for design of time-resolved experiments will be measured. The goal is to deliver a solution for millisecond time-resolved studies of protein dynamics that can be used by anyone in their home lab on any protein crystal system that can host the reactions and/or structural changes of interest. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.