PICOSECOND TIME-RESOLVED WAXS OF PROTEINS IN SOLUTION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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X射线脉冲探测并记录其衍射图样，研究血红蛋白（Hb）T/R跃迁的动力学。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。我们的目标是使用血红蛋白的WAXS模式作为一个“指纹”，可以匹配到已知的X射线结构，以跟踪血红蛋白的哪一部分是在T和R状态作为时间的函数。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之一，我们计划进行比较研究与肌红蛋白（Mb）。我们预期Mb会有与Hb相同的超快三级反应，但它缺少四级跃迁。 作为一个更简单的模型系统的血红蛋白，我们计划研究的二聚体血红蛋白的Scapharca inaequalivis（HbI）。HbI只有三个不同的连接状态，相比之下，在血红蛋白的情况下，12。进一步的蛋白质，我们计划研究的是神经红蛋白，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），细菌的光传感器。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.