Measuring Protein Topology in Conformationally Heterogeneous Mixtures by Top-Down Hydroxyl Radical Protein Footprinting

在化学测量和成像项目的支持下，密西西比大学的约书亚夏普博士和他的团队正在开发分析工具，以提高确定生物分子（包括蛋白质）三维形状的能力-这是在分子水平上理解生物学所必需的信息。 特别令人感兴趣的是涉及在多种形状之间波动的蛋白质的具有挑战性的应用。 夏普集团正在开发强大的新方法来表征形状共存的解决方案。 作为这项技术开发的一部分，他们正在为博士后研究助理和本科生通过少数民族外展计划在密西西比大学招募的实践研究培训机会。该项目正在开发的方法，可以提供新的见解结构异质蛋白质，包括（但不限于）构象动态蛋白质和蛋白质聚集中遇到的。 该方法还可以阐明蛋白质序列变异和翻译后修饰的结构后果。该方法结合了高分辨率羟基自由基蛋白质足迹（HR-HPF）与自上而下的串联质谱（MS/MS）分析的进展。 在HRPF中，氨基酸侧链与原位产生的羟基自由基的反应速率用于评估侧链？的溶剂可及性。 以前的工作表明，不同的非共价构象共存的混合物经历不同的氧化量时，受到高分辨力。 然而，“自下而上”的HR-HRPF目前只检测所有构象异构体的总体平均值;结构细节在消化和自下而上分析时丢失。 通过使用HR-HRPF自上而下的分析，可以根据整体修饰的程度分离构象异构体。 该技术的发展集中在两个系统的可控构象变化。 野生型CXCL 4（四聚体）与CXCL 4（二聚体）的K50 E突变体的混合物用于测试在溶液中独立表征二聚体和四聚体的能力，使用突变体的同位素标记以按质量分离构象异构体并验证来自未标记样品的结果。 第二个测试系统探测泛素的未折叠和折叠状态的不同羟基自由基反应性，以基于每种蛋白质中发生的氧化事件的数量来区分构象异构体。 从这个项目中获得的知识将有利于在结构生物学中一些最困难的问题的前沿工作的研究人员-构象的动态群体的详细表征，包括聚集，固有的无序蛋白质和蛋白质错误折叠/展开。该技术还将在分析蛋白质序列变异和蛋白质翻译后修饰的结构后果中发挥重要作用。

With support from the Chemical Measurement and Imaging Program, Dr. Joshua Sharp and his group at the University of Mississippi are developing analytical tools to advance the ability to determine the three-dimensional shape of biomolecules (including proteins) - information essential for understanding biology at the molecular level. Of particular interest are challenging applications involving proteins that fluctuate between multiple shapes. The Sharp group is developing powerful new methods to characterize shapes coexisting in solution. As part of this technology development, they are incorporating hands-on research training opportunities for postdoctoral research associates and undergraduate students recruited via minority outreach programs at the University of Mississippi.This project is developing methodology that can provide new insights into structurally heterogeneous proteins, including (but not limited to) conformationally dynamic proteins and those encountered in protein aggregation. The methods can also illuminate the structural consequences of protein sequence variations and post-translational modifications. The approach combines advances in high resolution hydroxyl radical protein footprinting (HR-HRPF) with top-down tandem mass spectrometry (MS/MS) analyses. In HRPF, the rate of reaction of amino acid side chains with hydroxyl radicals generated in situ is used to assess the side chain?s solvent accessibility. Previous work has shown that different non-covalent conformations co-existing in a mixture experience differing amounts of oxidation when subjected to HRPF. However, "bottom-up" HR-HRPF as currently practiced only detects an overall average of all conformers; structural details are lost upon digestion and bottom-up analysis. By using top-down analyses with HR-HRPF, conformers can be separated based on the extent of overall modification. Development of this technology is focusing on two systems with controllable conformational changes. Mixtures of wild-type CXCL4 (a tetramer) with the K50E mutant of CXCL4 (a dimer) are being used to test the ability to characterize the dimer and tetramer independently in solution, using isotopic labeling of the mutant to separate the conformers by mass and verify results from the unlabeled samples. A second test system probes the differential hydroxyl radical reactivity of the unfolded and folded states of ubiquitin to differentiate the conformers based on the number of oxidation events that occur in each protein. The knowledge derived from this project will benefit researchers working at the forefront of some of the most difficult problems in structural biology - the detailed characterization of dynamic populations of conformations including aggregation, inherently disordered proteins, and protein misfolding/unfolding. This technology would also play a powerful role in the analysis of the structural consequences of protein sequence variants and protein post-translational modifications.