RADICAL FOOTPRINTING BY SUB-MICROSECOND ELECTRON BEAM PULSE

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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. Hydroxyl radical footprinting is a new method for studying protein structure by examining differences in the rate of hydroxyl radical-protein sidechain reaction between a free protein and a protein-carbohydrate complex, and correlating changes in the rate of oxidation with changes in solvent accessibility. In order to probe only the native structure of the protein-carbohydrate complex, one must either strictly limit the reaction to one oxidation event per protein-carbohydrate complex, or limit the timescale of the reaction to complete faster than large scale protein conformational changes can occur. It has previously been shown that laser-induced photolysis of hydrogen peroxide can result in heavy oxidation that can be controlled to be completed on the sub-microsecond timescale. Unfortunately, this protocol requires high concentrations of hydrogen peroxide, which is capable of directly oxidizing proteins by both metal-catalyzed and metal-free mechanisms, on very fast timescales. We are working to develop a technique to oxidize proteins on a sub-microsecond timescale without the use of a precursor oxidant by radiolysis of water using an electron pulse from a Van de Graaff accelerator.