Exploring tyrosine redox chemistry in model proteins

描述（由申请人提供）：氨基酸基自由基参与生物体的一系列生产和破坏过程。功能性的，通常是高度控制的，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、稳定和远程迁移的基本热力学参数（即还原电位和pKA值）的实验表征仍然是初级的。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的反应性和热力学“热”自由基状态是极具挑战性的。我的博士研究项目的重点是扩展和探索一个专门用于研究蛋白质自由基化学的结构良好的模型蛋白库。这些模型蛋白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。利用这些模型系统，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。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值联系起来。这些性质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氧化还原偶联质子反应的函数来研究。在实验上，两种主要的方法是用于电化学分析的脉冲伏安法和用于结构研究的高分辨率蛋白质核磁共振波谱法。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。特异性目标2涉及酪氨酸自由基诱导的玻尔效应的表征，即蛋白质支架内氧化还原偶联的酸/碱反应。最后，Specific Aim 3包括努力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，如泛素。获得的数据将在自然系统、小分子溶液研究和理论工作之间提供必要和独特的联系。提出的研究具有很高的潜力，为天然氨基酸自由基系统的热力学讨论和机制模型的评估提供直接相关的数据。拟议研究的更广泛、更长期的目标是建立一个平台，在这个平台上分析自然系统中功能和功能失调的氨基酸自由基的特性。

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.