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

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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.