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Joshua Sharp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多种形状之间波动的蛋白质的挑战性应用。夏普小组正在开发强大的新方法来表征共存于溶液中的形状。作为这项技术发展的一部分，他们正在为通过密西西比大学少数民族拓展项目招募的博士后研究助理和本科生提供实践研究培训机会。该项目正在开发一种方法，可以为结构异质蛋白质提供新的见解，包括（但不限于）构象动态蛋白质和蛋白质聚集中遇到的蛋白质。这些方法还可以阐明蛋白质序列变化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足迹（HR-HRPF）和自上而下串联质谱（MS/MS）分析的进展。在HRPF中，氨基酸侧链与原位生成的羟基自由基的反应速率用于评估侧链？S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数量的氧化时，受到HRPF。然而，目前实践的“自下而上”HR-HRPF仅检测所有符合性的总体平均值；结构细节在消化和自下而上的分析中丢失了。通过使用自顶向下的HR-HRPF分析，可以根据整体修改的程度来分离构象。该技术的发展主要集中在两个具有可控构象变化的体系上。野生型CXCL4（四聚体）与CXCL4（二聚体）的K50E突变体的混合物被用来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通过质量分离构象，并验证未标记样品的结果。第二个测试系统探测泛素的未展开和折叠状态的不同羟基自由基反应活性，以根据每个蛋白质中发生的氧化事件的数量来区分构象。从这个项目中获得的知识将有利于研究结构生物学中一些最困难问题的前沿研究人员——包括聚集、固有无序蛋白质和蛋白质错误折叠/展开在内的动态构象群体的详细特征。这项技术也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方面发挥重要作用。

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.