RADICAL FOOTPRINTING BY SUB-MICROSECOND ELECTRON BEAM PULSE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 羟基自由基足迹是一种研究蛋白质结构的新方法，它通过检测游离蛋白质和蛋白质-碳水化合物复合体之间的羟基自由基-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联系起来。为了只探测蛋白质-碳水化合物复合体的天然结构，必须将反应严格限制为每个蛋白质-碳水化合物复合体一个氧化事件，或者限制反应的完成时间快于蛋白质构象的大规模变化。以前已经表明，激光诱导的过氧化氢光解可以导致严重的氧化，可以控制在亚微秒的时间尺度上完成。 不幸的是，这一方案需要高浓度的过氧化氢，它能够在非常快的时间尺度上通过金属催化和非金属催化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Van de Graaff加速器的电子脉冲对水进行辐射分解，在亚微秒的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.