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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们旨在用时间分辨广角X射线散射技术研究血红蛋白(Hb)从R到T四元结构转变的动力学。当从肺向组织输送氧气时，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中变构调节的最著名的例子。静态结晶学对这两种状态的结构都很清楚，但这种结构转变的速度仍然存在争议。以前对这种结构转变的时间分辨光谱研究是基于局部光谱标记的，并且只提供关于整体蛋白质构象的间接信息。我们的目标是使用Hb的TR-WAXS模式作为能够与已知X射线结构匹配的“指纹”，从而能够跟踪T和R态的随时间变化的布居。蛋白质样品是~1 mm的碳氧基血红蛋白(HbCO)溶液，将被密封在X射线毛细管中，并安装在电动线性工作台上，该工作台的平移与激光和X射线脉冲同步。我们使用CO作为O2的替代品，因为它可以高效地光解。激光脉冲(泵浦)之后是X射线脉冲(探头)，该脉冲由高速斩波器从同步加速器脉冲序列中分离出来。泵浦脉冲和探测脉冲之间的时间延迟由可编程的时间延迟设置，并且广角X射线散射图案由面探测器记录。由于CO可逆地与Hb结合，泵浦-探测循环可以重复数千次，而不会使样品降级。我们将在大约90年的时间内获得TR-WAXS模式。我们还将改变光解强度，以评估四元结构转变的速度对脱色程度的敏感程度。

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.