ULTRAFAST MEASUREMENTS OF PROTEIN FOLDING USING SAXS

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We propose the application of the rapid continuous flow technique to make small angle x-ray scattering (SAXS) measurements of the early stages of protein folding in the 1-50 ms time range. The conventional stopped-flow SAXS technique results in a time resolution of >50 ms and a dead time of 10 ms. Using a jet-mixer, the dead time will be reduced to 1 ms and a time resolution of 1 ms should be achieved. Using the combination of SAXS and the rapid continuous mixing technique, we will measure an unfolded protein's rate of compaction when it is suddenly placed under native conditions. SAXS is the only known time-resolved technique which gives overall size and shape indication of the conformational state of a protein. SAXS data may be used to determine the radius of gyration and to obtain shape information which may be inferred from the Kratky plot and the pair distribution function. The earliest data points for the stopped-flow technique usually show a relatively compact structure as compared to the unfolded state. With the dramatically improved time resolution and reduced dead time, we will be able to study the rate of compaction thoroughly at the early stages of refolding.