Time-Resolved Protein Structure from Non-Crystallized Molecular Ensembles

光激发下蛋白质结构的快速变化对自然界中许多重要过程的阐明有影响，例如光合作用和蛋白质中的其他光诱导化学反应。伴随蛋白质结晶的变化可以通过时间分辨X射线晶体学技术进行实验，该技术涉及布拉格点的测量和索引。然而，并不是所有的蛋白质（特别是膜蛋白）参与许多关键的生命过程，形成晶体。此外，晶体中的蛋白质所经历的反应可能无法反映生命系统中蛋白质的反应。因此，能够通过实验研究溶液中蛋白质的光诱导化学反应将是一个重大突破。本项目的目的是开发一种有效的理论方法，用于从泵浦探测实验中提取结构信息，该实验旨在跟踪光激发后单分子的结构变化。该项目将通过测量直线加速器相干光源（LCLS）处的X射线自由电子激光（XFEL）的漫射衍射图案来证明这种能力。在光激发之前和之后，将从含有相同蛋白质集合的液滴样品获得衍射图案。光激发光束将是与XFEL脉冲同步的激光，其将在分子被XFEL光束询问之前的短时间内激发分子。对于随机取向的分子系综，通过评价衍射数据的角度相关性，将开发利用来自分子系综的较大信号的能力。这一想法将首先证明从计算机模拟的数据，这种衍射图案，然后与测量实验衍射图案的演示。后一项工作将涉及与亚利桑那州立大学的John Spence教授的合作，他具有在加利福尼亚州斯坦福大学的直线加速器相干光源（LCLS）使用XFEL进行蛋白质结构工作的经验。更广泛的影响在密尔沃基大学本科生研究机会计划（UROP）的帮助下招募的本科生将被纳入研究工作。尖端的结构生物学技术将被引入高中课堂的K-12教师招募到校园的帮助下，由威斯康星大学密尔沃基分校的NSF资助的教师研究经验（RET）计划。教师将首先与主要研究人员一起参与研究工作，并将他们所学的知识转移到课堂上。一个新设计的网页将提高研究工作的能见度和影响。

Intellectual MeritFast changes in the structure of a protein on photoexcitation have implications for the elucidation of many important processes in nature, such as photosynthesis and other photo-induced chemical reactions in proteins. Changes accompanying protein crystallization can be experimentally followed by the technique of time-resolved X-ray crystallography, which involves the measurement and indexing of Bragg spots. However, not all proteins (particularly membrane proteins) involved in many of the crucial processes of life, form crystals. In addition, reactions undergone by proteins in a crystal may not mirror those of proteins in living systems. As such, an ability to study experimentally photo-induced chemical reactions of proteins in solution would constitute a significant breakthrough. The objective of this project is to develop a valid theoretical method for extracting structural information from a pump-probe experiment designed to follow structural changes of single molecules immediately after photoexcitation. This project will demonstrate this capability through measurements of diffuse diffraction patterns from an X-ray free electron laser (XFEL) at the Linac Coherent Light Source (LCLS). The diffraction pattern will be obtained from a sample of liquid droplets, containing ensembles of identical proteins, before and after photoexcitation. The photoexciting beam will be a laser, synchronized with the XFEL pulses, which will excite the molecules a short time before they are interrogated by the XFEL beam. The ability to take advantage of larger signals from ensembles of molecules will be developed, for ensembles of randomly oriented molecules, by the evaluation of angular correlations of the diffraction data. The idea will be demonstrated initially with data from computer simulations of such diffraction patterns and followed by demonstrations with measured experimental diffraction patterns. The latter work will involve collaboration with Prof. John Spence of Arizona State University, who has experience of protein structure work with the XFEL at the Linac Coherent Light Source (LCLS) in Stanford, CA. Broader ImpactsUndergraduates recruited with the help of the UW-Milwaukee's undergraduate research opportunity program (UROP) will be integrated into the research efforts. Cutting-edge techniques of structural biology will be introduced into high-school classrooms with the help of K-12 teachers recruited to the campus by the UW-Milwaukee's NSF-funded research experience for teachers (RET) program. Teachers will first participate in research efforts with the principal investigators and transfer what they learn into their classrooms. A newly designed web-page will increase the visibility and impact of the research efforts.