PICOSECOND TIME-RESOLVED WAXS OF PROTEINS IN SOLUTION

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们的目标是采用皮秒时间分辨广角散射（WAXS）技术来研究蛋白质在溶液中的动力学。该技术可用于天然光敏的蛋白质或可使其光敏并在光激发时经历可逆光循环的蛋白质。我们用激光脉冲激发蛋白质溶液样品，用延时的X射线脉冲探测它，并记录它的衍射图案。 我们计划研究血红蛋白（Hb）的T/R转换的动力学。当将氧气从肺输送到组织时，Hb在高亲和力和（R）和低亲和力（T）构象之间切换。这两种状态的结构是众所周知的静态晶体学，但与过渡发生的速度仍有很大的争议。文献中报道的基于光谱技术的动态研究仅基于局部光谱标记，并且仅给出关于整体蛋白质构象的间接信息。我们的目标是使用Hb的WAXS模式作为“指纹”，可以与已知的X射线结构相匹配，以跟踪Hb的哪一部分作为时间的函数处于T和R状态。我们使用HbCO作为HbO 2的替代品，因为它可以被高效的激光闪光光解。CO在几毫秒内重新结合到Hb上。 由于血红蛋白显示出超快的三级响应的光解和时间延迟的四级，我们计划进行比较研究与肌红蛋白（Mb）。我们预期Mb的超快三级反应与Hb相同，但缺少四级跃迁。 作为一个更简单的模式系统的血红蛋白，我们计划研究的二聚血红蛋白的Scapharca inaequalivis（HbI）。HbI只有三种不同的连接状态，而Hb有十二种。 我们计划进一步研究的蛋白质是脑红蛋白，肌红蛋白样蛋白，在大脑中发现，作为氧传感器和光敏黄蛋白（PYP）细菌光传感器。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We aim to employ the technique of picosecond time-resolved wide-angle scattering (WAXS) to investigate the dynamics of proteins in solution. This technique can be employed on proteins that are naturally light-sensitive or can be made light sensitive and undergo a reversible photocycle when excited by light. We use a laser pulse to excite a sample of protein solution probe it with a time-delayed X-ray pulse and record its diffraction pattern. We plan to investigate the kinetics of the T/R transition of hemoglobin (Hb). When transporting oxygen from the lungs to the tissues Hb switches between a high affinity and (R) and low affinity (T) conformation. The structure of both state are well known by static crystallography but the rate with which the transition occurs in still much in debate. Dynamic studies based on spectroscopic techniques reported in the literature are based only local spectroscopic markers and give only give indirect information about the overall protein conformation. We aim to use the WAXS pattern of Hb as an 'fingerprint' that can be matched to known X-ray structures to track which fraction of Hb is in T and R state as function of time. We use HbCO as substitute for HbO2 because it can be photolyzed with a laser flash with high efficiency. The CO rebinds to the Hb within a few milliseconds. Since Hb shows both an ultrafast tertiary response to the photolysis and a time delayed quaternary one we plan to perform a comparative study with myoglobin (Mb). We would expect in the case of Mb the same ultrafast tertiary response as Hb but it lacks a quaternary transition. As a simpler model system for Hb we plan to study the dimeric hemoglobin of Scapharca inaequivalvis (HbI). HbI has only three different ligation states compared to twelve in the case of Hb. Further proteins we plan to investigate are neuroglobin myoglobin-like protein found in the brain acting as an oxygen sensor and the Photoactive Yellow Protein (PYP) bacterial photosensor.