QUANTITATION OF OXIDATION-INDUCED CONFORMATIONAL CHANGES BY 2ND ORDER REACTIONS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recently, a method for hydroxyl radical footprinting using flash fast photooxidation of proteins (FPOP) was published that claims to complete the oxidation chemistry faster than the protein can undergo a significant oxidation-induced unfolding. Unfortunately, the methods for verifying this claim were indirect in nature, by comparing sites of oxidation with published high resolution structures. In order to verify that the FPOP method can complete oxidation chemistry faster than the protein can unfold, we are developing a method for detecting and quantitating the amount of oxidation-induced unfolding that occurs during a second order hydroxyl radical footprinting experiment using analysis of the intact protein oxidation products.