Grant: Research Tools and Instruments - Category 1 (<$150,000)

The question how and why unfolded proteins can spontaneously fold to their biologically active conformations remains one of the major unsolved problems in biological chemistry. One particularly important bottleneck is the lack of information regarding the very earliest stages of folding. These ultra-rapid processes occur on a sub-millisecond time scale, which makes it exceedingly challenging to characterize the structures of early folding intermediates. Past efforts in this area have largely relied on spectroscopic probes that are unable to supply detailed insights into protein conformations. This proposal describes a novel strategy that will for the first time provide "snapshots" of protein structures during the very earliest stages of folding. Ultra-rapid diffusive mixing will expose denatured polypeptide chains to native conditions, thereby triggering the folding process. An excimer laser will then produce OH radicals by photolysis of H2O2, resulting in microsecond covalent labeling of the protein at well defined time points. Importantly, labeling only occurs in solvent-accessible regions, whereas folded segments are protected. Analyses of the labeling pattern by mass spectrometry will then result in highly detailed solvent exposure data which reflect the structural changes during folding. Our group was the first to demonstrate the viability of such an approach in 2009, albeit on relatively slow time scales. Unfortunately, the limited specifications of our existing laser currently preclude the application of this strategy to ultra-rapid folding studies. We therefore propose the acquisition of a new 750 Hz laser that will allow an extension of our work to the sub-millisecond range. This proposal represents a unique opportunity; it will allow our laboratory to pioneer the exploration of ultra-rapid folding events with hitherto unobtainable structural resolution. The resulting insights will enhance the general understanding of biological self-assembly, as well as aberrant side reactions that can lead to disease states. These research activities will provide superb opportunities for HQP training. Failure to secure funding for this equipment would be a severe setback for our research program.