This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Protein-carbohydrate interactions of various types are difficult to characterize by standard high-resolution structural techniques. 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. One great difficulty in hydroxyl radical footprinting is the issue of oxidation-induced conformational changes; after an amino acid becomes oxidized, the resulting change in the physical properties of the amino acid causes changes of varying magnitude in the structure. 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.
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