NANOSECOND TEMPERATURE-JUMP SAXS

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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. There is a worldwide effort to try to predict protein folds from their sequences. To supplement these computational efforts, a number of techniques are used to identify the intermediates in this process and measure the kinetics of the changes between these intermediate states. The time resolution of these methods is key, as it limits their utility in identifying protein folding intermediates. Temperature-jump techniques have received increasing attention because of the availability of high-power lasers that can uniformly and suddenly increase the water temperature of a small sample tens of degrees Celsius without the use of added dyes or other absorbers. This method has been combined with several time-resolved optical techniques to study protein folding, catalysis, and reaction kinetics. Unfortunately, visible light probes such as fluorescence or absorption spectroscopies yield a limited amount of local structural information. In contrast, SAXS provides information on global shape changes of a protein in solution. However, present methods are likely to remain limited to ~100 microsecond temporal resolution and have therefore been unable to measure the earliest and fastest folding events, such as those in involved in downhill protein folding or hydrophobic collapse where the entire process may take only a few tens of microseconds or less. We intend to circumvent the inherent deadtime and signal-to-noise limitations of existing techniques to reach sub-microsecond timescales in solution SAXS. In the past year we have estimated exposure times, built instrumentation, and fully characterized the static behavior of the protein systems we will be using for future time-resolved measurements.