RADICAL FOOTPRINTING BY SUB-MICROSECOND ELECTRON BEAM PULSE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 羟基自由基足迹法是一种研究蛋白质结构的新方法，通过检测游离蛋白质和蛋白质-碳水化合物复合物之间羟基自由基-蛋白质侧链反应速率的差异，并将氧化速率的变化与溶剂可及性的变化相关联。 为了仅探测蛋白质-碳水化合物复合物的天然结构，必须将反应严格限制为每个蛋白质-碳水化合物复合物一次氧化事件，或者将反应的时间尺度限制为比大规模蛋白质构象变化可能发生的更快完成。 先前已经表明，过氧化氢的激光诱导光解可以导致可以控制在亚微秒时间尺度上完成的重氧化。 不幸的是，该方案需要高浓度的过氧化氢，其能够在非常快的时间尺度上通过金属催化和无金属机制直接氧化蛋白质。 我们正在努力开发一种技术，通过使用来自货车德格拉夫加速器的电子脉冲来辐解水，在亚微秒时间尺度上氧化蛋白质，而不使用前体氧化剂。

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. 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.