PICOSECOND TIME-RESOLVED WAXS OF PROTEINS IN SOLUTION

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 我们的目标是使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。这项技术可以应用于自然对光敏感的蛋白质，或者可以使其对光敏感，并在光激发时经历可逆光循环。我们用激光脉冲激发蛋白质溶液的样品，用延迟的X射线脉冲探测它，并记录它的衍射图。 我们计划研究血红蛋白(Hb)T/R转变的动力学。当氧气从肺部输送到组织时，Hb在高亲和力构象和(R)构象和低亲和力(T)构象之间切换。静态结晶学对这两种状态的结构都很清楚，但这种转变发生的速度仍然有很大争议。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模式作为可以与已知X射线结构匹配的“指纹”，以跟踪Hb的哪一部分处于T和R状态随时间的变化。我们使用HbCO作为HBO2的替代品，因为它可以用激光闪光灯高效地进行光解。CO在几毫秒内重新结合到HB上。 由于Hb对光解表现出超快的三级反应和延时的四级反应，我们计划与肌红蛋白(Mb)进行比较研究。在Mb的情况下，我们预计会有与Hb相同的超快第三级响应，但它缺乏第四级跃迁。 作为研究Hb的更简单的模型系统，我们计划研究Scapharca inaequvalvis(HBI)的二聚体血红蛋白。HbI只有3种不同的连接状态，而Hb有12种不同的连接状态。 我们计划进一步研究的蛋白质是在大脑中发现的作为氧传感器的脑红蛋白肌红蛋白样蛋白和光活性黄色蛋白(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.