TIME-RESOLVED WAXS STUDY OF THE T/R TRANSITION OF HEMOGLOBIN

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 利用时间分辨广角X射线散射（TR-WAXS）技术研究了血红蛋白（Hb）从R到T四级结构转变的动力学过程。当将氧气从肺输送到组织时，Hb从高亲和力R构象转换为低亲和力T构象，从而使其能够以非常高的效率将O2输送到组织。这种结构-功能关系可能是蛋白质变构调节的最著名的例子。这两种状态的结构已为静态晶体学所熟知，但这种结构转变的速率仍存在争议。以前的时间分辨光谱研究，这种结构的转变是基于本地的光谱标记，只提供间接信息的整体蛋白质构象。我们的目标是使用Hb的TR-WAXS模式作为“指纹”，可以与已知的X射线结构相匹配，因此可以跟踪T和R状态的时间依赖性种群。将蛋白质样品（约1 mM的碳氧血红蛋白（HbCO）溶液）密封在X射线毛细管中，并安装在电动线性载物台上，该载物台的平移与激光和X射线脉冲同步。我们使用CO作为O2的替代物，因为它可以高效光解。激光脉冲（泵浦）之后是X射线脉冲（探测），该脉冲通过高速斩波器与同步加速器脉冲串隔离。泵浦和探测脉冲之间的时间延迟由可编程时间延迟设置，广角X射线散射图案由区域检测器记录。因为CO可逆地结合到Hb，所以泵-探测循环可以重复数千次而不会降解样品。我们将在大约90年的时间内获得TR-WAXS图案。我们还将改变光解强度，以评估四级结构转变的速率对脱配程度的敏感程度。

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 investigate the kinetics of the R to T quaternary structure transition of hemoglobin (Hb) using the technique of time-resolved wide-angle X-ray scattering (TR-WAXS). When transporting oxygen from the lungs to the tissues, Hb switches from a high-affinity R to a low-affinity T conformation, thereby enabling it to deliver O2 to the tissues with very high efficiency. This structure-function relationship is perhaps the best-known example of allosteric regulation in proteins. The structure of both states are well known by static crystallography, but the rate of this structure transition in still being debated. Prior time-resolved spectroscopic studies of this structure transition are based on local spectroscopic markers and provide only indirect information about the overall protein conformation. We aim to use the TR-WAXS pattern of Hb as a 'fingerprint' that can be matched to known X-ray structures and can therefore track the time-dependent population of the T and R states. The protein sample, ~1-mM solution of carboxy hemoglobin (HbCO), will be sealed in an X-ray capillary and mounted on a motorized linear stage whose translation is synchronized to the laser and X-ray pulses. We use CO as a surrogate for O2 because it can be photolyzed with high efficiency. The laser pulse (pump) is followed by an X-ray pulse (probe) that is isolated from the synchrotron pulse train by a high-speed chopper. The time delay between the pump and probe pulses is set by a programmable time delay, and the wide-angle X-ray scattering pattern is recorded by an area detector. Because the CO binds to the Hb reversibly, the pump-probe cycle can be repeated thousand of times without degrading the sample. We will obtain TR-WAXS patterns over approximately 9 decades of time. We will also vary the photolysis intensity to assess the extent to which the rate of the quaternary structure transition is sensitive to the degree of deligation.