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: 8231264
• 负责人: Melissa Martinez-Rivera
• 金额: $ 4.22万
• 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 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8231264
• 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. PUBLIC HEALTH RELEVANCE: Amino-acid radicals play essential roles in biological processes, such as DNA synthesis and repair, hormone synthesis, carbohydrate metabolism, cell detoxification reactions, and energy transduction. Due to the high reactivity of these species and level of complexity of their protein hosts, the systematic study of the redox properties of amino-acid radicals as a function of protein structure has not been successful. For this reason, we have developed a series of model protein scaffolds with the characteristics necesary to investigate the relation between the thermodynamics of amino-acid redox chemistry and the surrounding protein environment.

描述（由申请人提供）：基于氨基酸的自由基参与生物体中的一系列生产性和破坏性过程。功能性且通常高度受控的自由基化学发生在使用氨基酸作为催化活性氧化还原辅助因子的酶中。相比之下，众所周知，氧化应激条件会产生一系列不受控制的、对生物体潜在有害的蛋白质自由基反应。尽管已知的氨基酸自由基酶的数量和与功能失调的氨基酸自由基化学相关的病理条件不断增加，但涉及蛋白质自由基形成、稳定和长程迁移的基本热力学参数（即还原电位和 pKA 值）的实验表征仍然处于初级阶段。这种情况反映了一个简单的事实，即在自然系统中研究特征性反应性和热力学“热”自由基态非常具有挑战性。我博士学位的重点。研究项目是扩展和探索专门用于研究蛋白质自由基化学的结构良好的模型蛋白质库。这些模型蛋白质包含感兴趣的自由基位点以及有助于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绘制氨基酸自由基的热力学性质作为蛋白质结构特征的函数。所提出的实验研究的总体目标是将特定的结构特征与酪氨酸自由基的还原电位和 pKA 值相关联。这些特性将作为蛋白质支架内静电相互作用、氢键相互作用、溶剂暴露程度和氧化还原耦合质子反应的函数进行研究。在实验上，将使用的两种主要方法是用于电化学分析的脉冲伏安技术和用于结构研究的高分辨率蛋白质核磁共振波谱。具体目标 1 包括探索自由基辅助因子-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 涉及酪氨酸自由基诱导的玻尔效应的表征，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 涉及扩展我们的自由基模型蛋白家族，以包括天然来源的模型蛋白，例如泛素。获得的数据将为自然系统、小分子溶液研究和理论工作之间提供重要且独特的联系。拟议的研究很有可能提供与天然氨基酸自由基系统的热力学讨论和机械模型评估直接相关的数据。拟议研究的更广泛、更长期的目标是建立一个平台来分析自然系统中功能性和功能失调的氨基酸自由基的特性。 公共健康相关性：氨基酸自由基在 DNA 合成和修复、激素合成、碳水化合物代谢、细胞解毒反应和能量转导等生物过程中发挥着重要作用。由于这些物种的高反应性及其蛋白质宿主的复杂性，对氨基酸自由基的氧化还原特性作为蛋白质结构的函数的系统研究尚未成功。为此，我们开发了一系列模型蛋白质支架，其具有研究氨基酸氧化还原化学热力学与周围蛋白质环境之间关系所需的特性。