Exploring tyrosine redox chemistry in model proteins

探索模型蛋白质中的酪氨酸氧化还原化学

• 批准号: 8401128
• 负责人: Melissa Martinez-Rivera
• 金额: $ 1.76万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8401128
• 关键词: Acids; Amino Acids; Biochemical; Biological; Biological Models; Biological Process; Bohr effect; Cells; Characteristics; Chemistry; Coupled; Cysteine; DNA Repair; DNA biosynthesis; Data; Dependence; Doctor of Philosophy; Drug Metabolic Detoxication; Electrostatics; Engineering; Environment; Enzymes; Evaluation; Family; Generations; Goals; Histidine; Hormones; Hydrogen Bonding; Imidazole; Libraries; Life; Link; Literature; Maps; Measurement; Methods; Micrococcal Nuclease; Modeling; Mutagenesis; Nature; Organism; Oxidation-Reduction; Oxidative Stress; Oxygen; Phenols; Physiologic pulse; Play; Positioning Attribute; Process; Property; Protein Binding; Protein NMR Spectroscopy; Proteins; Protons; Reaction; Relative (related person); Research; Research Project Grants; Research Proposals; Resolution; Role; Scaffolding Protein; Series; Side; Site; Solutions; Solvents; Structural Protein; Structure; System; Techniques; Theoretical Studies; Thermodynamics; Tyrosine; Ubiquitin; Variant; Water; Work; aqueous; base; carbohydrate metabolism; carcinogenesis; chemical property; cofactor; design; interest; migration; oxidation; protein expression; protein function; protein structure function; public health relevance; research study; small molecule; tyrosine radical

DESCRIPTION (provided by applicant): Amino-acid based radicals are involved in a range of productive and destructive processes in living organisms. Functional, and typically highly controlled, radical chemistry occurs in enzymes that use amino acids as catalytically active redox cofactors. In contrast, oxidative stress conditions are well known to generate a range of uncontrolled, and for the organism, potentially harmful protein radical reactions. Despite the fact that the number of known amino-acid radical enzymes and pathological conditions linked to dysfunctional amino-acid radical chemistry continuously increases, experimental characterization of basic thermodynamic parameters (i.e. reduction potentials and pKA values) involved in protein radical formation, stabilization, and long-range migration is still rudimentary. This situation reflects the simple fact that the characteristically reactive and thermodynamically "hot" radical state is highly challenging to study in the natural systems. The focus of my Ph.D. research project is to extend and explore a library of well-structured model proteins specifically made to study protein radical chemistry. These model proteins contain the radical site of interest as well as features that will facilitate the biophysical characterization of the radical and its surounding protein environment. Using these model systems, we aim to systematically map the thermodynamic properties of amino-acid radicals as a function of protein structural features. The overall objective with the proposed experimental studies is to correlate specific structural features with the reduction potentials and pKA values of tyrosine radicals. These properties will be studied as a function of electrostatic interactions, hydrogen-bonding interactions, degree of solvent exposure and redox-coupled protonic reactions within the protein scaffold. Experimentaly, the two main methods that will be utilized are pulsed voltammetry techniques for electrochemical analyses and high- resolution protein NMR spectroscopy for structural studies. Specific Aim 1 includes work to probe radical cofactor-solvent interactions and the influence of these interactions on the redox properties of tyrosine. Specific Aim 2 involves the characterization of tyrosine radical induced Bohr effects i.e. redox-coupled acid/base reactions within the protein scaffold. Finally, Specific Aim 3 involves efforts to extend our family of radical model proteins to include model proteins of natural origin, such as ubiquitin. The obtained data will provide an essential and unique link between the natural systems, small-molecule solution studies and theoretical work. The proposed studies have a high potential to provide data that are directly relevant to thermodynamic discussions of natural amino-acid radical systems and evaluations of mechanistic models. The broad, more long-term objective with the proposed studies is to build a platform on which to analyze the properties of functional and dysfunctional amino-acid radicals in natural systems.

描述（由申请人提供）：基于氨基酸的自由基参与生物体的一系列生产和破坏过程。在使用氨基酸作为催化活性氧化还原辅因子的酶中，发生功能性且通常高度受控的自由基化学。相反，众所周知，氧化应激条件会产生一系列不受控制的，对生物体来说可能有害的蛋白质自由基反应。尽管事实上，已知的氨基酸自由基酶和病理条件的数量与功能失调的氨基酸自由基化学不断增加，实验表征的基本热力学参数（即还原电位和pKA值）参与蛋白质自由基的形成，稳定和远程迁移仍然是基本的。这种情况反映了一个简单的事实，即在自然系统中研究具有特征性反应性和热“热”的自由基状态是非常具有挑战性的。我博士研究的重点研究项目是扩展和探索一个结构良好的模型蛋白质库，专门用于研究蛋白质自由基化学。这些模型蛋白含有感兴趣的自由基位点以及将促进自由基及其周围蛋白质环境的生物物理表征的特征。使用这些模型系统，我们的目标是系统地映射作为蛋白质结构特征的函数的氨基酸自由基的热力学性质。与拟议的实验研究的总体目标是相关的具体结构特征与酪氨酸自由基的还原电位和pKA值。这些特性将作为静电相互作用，氢键相互作用，溶剂暴露程度和蛋白质支架内的氧化还原偶联质子反应的函数进行研究。实验上，将利用的两种主要方法是用于电化学分析的脉冲伏安法技术和用于结构研究的高分辨率蛋白质NMR光谱。具体目标1包括探测自由基辅因子-溶剂相互作用以及这些相互作用对酪氨酸氧化还原性质的影响。具体目标2涉及表征酪氨酸自由基诱导的玻尔效应，即蛋白质支架内的氧化还原偶联酸/碱反应。最后，具体目标3涉及的努力，以扩大我们的家庭激进的模型蛋白，包括模型蛋白的天然来源，如泛素。所获得的数据将提供自然系统，小分子溶液研究和理论工作之间的重要和独特的联系。拟议的研究有很大的潜力，提供数据，直接相关的热力学讨论天然氨基酸自由基系统和评价的机制模型。拟议研究的广泛，更长期的目标是建立一个平台，在此基础上分析自然系统中功能和功能失调的氨基酸自由基的性质。