Ultrafast time resolved protein X-ray crystallography

我们现在拥有所有的工具和方法，使我们能够以超快的时间分辨率实时观察蛋白质的结构分辨率，探测生物激活的基本过程。随着飞秒x射线自由电子激光源的可用性，现在在飞秒脉冲持续时间的硬x射线制度下工作，为获得对蛋白质超快结构过程的基本新见解提供了独特的机会。在这里，我提出了一个飞秒时间分辨的x射线晶体光敏蛋白晶体的光诱导反应和支持光谱研究的研究计划。这些实验的必要的前期工作是在斯坦福直线加速器相干光源（LCLS）收集的数据，从在家庭实验室进行的非线性跃迁和脉冲整形的超快光谱学研究到控制飞秒种群转移，以及基于测量源参数的晶体学数据质量的支持建模。除了主要目标是光活性黄色蛋白晶体的飞秒实验外，新的实验正在开发中，首先使用皮秒同步加速器源，以增加飞秒泵浦探针研究的现有选择，并专注于光切换荧光蛋白。

We now have all the tools and the methodology that allows us to watch proteins function with structural resolution in real time with ultrafast time resolution, probing fundamental processes in biological activation. With the availability of femtosecond X-ray Free Electron Laser sources which now operate in the hard X-ray regime with femtosecond pulse duration, unique opportunities arise to gain fundamental new insight into ultrafast structural processes in proteins. Here, I propose a research programme on femtosecond time-resolved X-ray crystallography of light induced reactions in light sensitive protein crystals and supporting spectroscopic studies. The necessary preliminary work for such experiments are provided in the form of data collected at the Stanford Linac Coherent Light Source (LCLS), from ultrafast optical spectroscopy investigations of non-linear transitions and pulse shaping to control femtosecond population transfer performed in the home laboratory, and from supporting modeling of crystallographic data quality based on measured source parameters. In addition to the primary target, which is a femtosecond experiment with Photoactive Yellow Protein crystals, novel experiments are being developed firstly using picosecond synchrotron sources, to add to the existing choices for femtosecond pump-probe studies and focuses on photoswitchable fluorescent proteins.