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

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们的目标是利用时间分辨广角 X 射线散射 (TR-WAXS) 技术研究血红蛋白 (Hb) 从 R 到 T 四级结构转变的动力学。当将氧气从肺部输送到组织时，血红蛋白从高亲和力的R构象转变为低亲和力的T构象，从而使其能够以非常高的效率将氧气输送到组织。这种结构-功能关系可能是蛋白质变构调节最著名的例子。这两种状态的结构通过静态晶体学是众所周知的，但这种结构转变的速率仍然存在争议。先前对该结构转变的时间分辨光谱研究基于局部光谱标记，并且仅提供有关整体蛋白质构象的间接信息。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 状态的时间依赖性群体。蛋白质样品，约 1 mM 的碳氧血红蛋白 (HbCO) 溶液，将被密封在 X 射线毛细管中，并安装在电动线性平台上，其平移与激光和 X 射线脉冲同步。我们使用 CO 作为 O2 的替代品，因为它可以高效地光解。激光脉冲（泵）后面跟随 X 射线脉冲（探头），X 射线脉冲通过高速斩波器与同步加速器脉冲串隔离。泵浦脉冲和探测脉冲之间的时间延迟由可编程时间延迟设置，并且广角 X 射线散射图案由区域探测器记录。由于 CO 可逆地与 Hb 结合，因此泵-探测循环可以重复数千次，而不会降解样品。我们将在大约 9 个十年的时间内获得 TR-WAXS 模式。我们还将改变光解强度，以评估四级结构转变速率对脱结程度的敏感程度。

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.