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

康奈尔大学获得了一个奖项，以开发新的技术和方法，用于对蛋白质的时间分辨研究。蛋白质是一种动态机器，它与其他分子相互作用，执行生命所需的无数功能。 使用X射线晶体学确定的蛋白质的三维结构揭示了它们的原子结构，提供了对它们如何发挥功能和发生故障的洞察，并为理解蛋白质功能的分子机制提供了基础。如果这些静态结构可以通过蛋白质的“分子电影”来补充，例如，当它们催化化学反应产生新分子时，就可以学到更多。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具有毫秒时间分辨率的“电影”，允许时间分辨晶体学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点。这项开发工作将涉及一个多元化的科学和工程团队，并努力招募将从所提供的实践和跨学科培训中受益最多的参与者。 如果这项工作取得成功，商业开发将有一条明确的道路，以最大限度地发挥该技术的影响。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。 使用自由电子激光X射线源的强大方法已经被开发用于由光或化学混合引发的生物分子反应的时间分辨研究，但是它们的应用是资源密集型的，并且不太适合广泛的科学和生物技术用途。 另一种方法已经被证明，其中在晶体中引发的反应在几十毫秒内通过超快速冷却淬灭，并且通过标准的邮寄冷冻扫描法从这些晶体中收集X射线数据。这种方法将反应的时间尺度与X射线数据收集的时间尺度分开，每个晶体产生更多有用的数据，并且最小化每个时间点结构测定所需的晶体数量。在这里，仪器和反应引发和快速冷却的方法将被开发，以减少可实现的时间分辨率为1毫秒，时间分辨实验设计的关键参数将被测量。 其目标是为毫秒级时间分辨的蛋白质动力学研究提供一个解决方案，任何人都可以在自己的家庭实验室中对任何蛋白质晶体系统进行研究，这些蛋白质晶体系统可以主持感兴趣的反应和/或结构变化。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值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.