Time-Resolved Protein Structure from Non-Crystallized Molecular Ensembles

智能价值在光激发下蛋白质结构的快速变化对阐明自然界中的许多重要过程有重要意义，如光合作用和蛋白质中其他光诱导的化学反应。时间分辨X射线结晶学技术可以通过实验来跟踪蛋白质结晶过程中的变化，这涉及到布拉格斑点的测量和索引。然而，并不是所有参与生命许多关键过程的蛋白质(尤其是膜蛋白)都会形成晶体。此外，蛋白质在晶体中发生的反应可能并不反映生命系统中蛋白质的反应。因此，能够在实验上研究蛋白质在溶液中的光诱导化学反应将是一项重大突破。本项目的目标是开发一种有效的理论方法，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立即发生的单分子的结构变化。该项目将通过在直线加速器相干光源(LCLS)上测量X射线自由电子激光器(XFEL)的漫射衍射图来展示这一能力。衍射图将从包含相同蛋白质系综的液滴样本中获得，在光激发前后。光激励光束将是与XFEL脉冲同步的激光，在分子被XFEL光束询问之前，这将在很短的时间内激发分子。对于随机取向的分子系综，通过评估衍射数据的角度相关性，将开发利用来自分子系综的更大信号的能力。这一想法将首先用计算机模拟的这种衍射图的数据来演示，然后用测量的实验衍射图进行演示。后一项工作将涉及与亚利桑那州立大学的John Spence教授合作，他在加利福尼亚州斯坦福的直线加速器相干光源(LCLS)的XFEL蛋白质结构工作方面拥有经验。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整合到研究工作中。结构生物学的尖端技术将在密尔沃基大学国家科学基金会资助的教师研究经验(RET)计划招聘到校园的K-12教师的帮助下引入高中课堂。教师将首先与主要调查人员一起参与研究工作，并将他们所学的知识转移到他们的课堂上。新设计的网页将提高研究工作的知名度和影响力。

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.