PICOSECOND TIME-RESOLVED WAXS OF PROTEINS IN SOLUTION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.