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

康奈尔大学被授予开发新的技术和方法，用于对作用中的蛋白质进行时间分辨研究。蛋白质是动态的机器，它与其他分子相互作用，执行生命所需的无数功能。用X射线结晶学测定蛋白质的三维结构揭示了它们的原子结构，为了解它们的功能和故障提供了洞察，为理解蛋白质功能的分子机制提供了基础。例如，当蛋白质催化化学反应产生新的分子时，如果这些静态结构可以被作用中的蛋白质的“分子电影”补充，那么我们可以了解更多。即将在这里开发的技术有望提供一种有效和高效的方法来制作这些时间分辨率为毫秒的“电影”，使时间分辨结晶学能够被更多不同机构的更多研究人员应用于更多的生物分子靶标。这项开发工作将吸引不同的科学和工程团队，努力招募将从所提供的实践和跨学科培训中受益最大的参与者。如果这项工作成功，就有一条明确的商业发展道路，可以最大限度地发挥这项技术的影响。这些研究工作得到了本科教育和教师培训方面的长期努力的补充。利用自由电子激光X射线源对光或化学混合引发的生物分子反应进行时间分辨研究已经发展出强大的方法，但它们的应用是资源密集型的，不太适合广泛的科学和生物技术用途。已经证明了另一种方法，即在晶体中启动的反应通过超快速冷却在数十毫秒内熄灭，并通过标准邮寄入的结晶术从这些晶体中收集X射线数据。这种方法将反应的时间刻度与X射线数据收集的时间刻度分开，每个晶体产生更多有用的数据，并最大限度地减少每个时间点确定结构所需的晶体数量。在这里，将开发用于反应引发和快速冷却的仪器和方法，以将可实现的时间分辨率降低到1ms，并将测量用于设计时间分辨实验的关键参数。其目标是为蛋白质动力学的毫秒时间分辨研究提供解决方案，任何人都可以在他们的家庭实验室中对任何可以承载感兴趣的反应和/或结构变化的蛋白质晶体系统使用该解决方案。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.