Understanding protein radicals

了解蛋白质自由基

• 批准号: 7667933
• 负责人: Cecilia Tommos
• 金额: $ 22.84万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-04 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7667933
• 关键词: Accounting; Address; Alanine; Amino Acids; Anions; Area; Biological Models; Biology; Camphor 5-Monooxygenase; Cations; Cells; Characteristics; Charge; Chemicals; Chemistry; Circadian Rhythms; Complex; Cysteine; Cytochrome c Peroxidase; Cytochromes; DNA biosynthesis; Development; Drug Metabolic Detoxication; Electron Nuclear Double Resonance; Electrons; Electrostatics; Engineering; Environment; Enzymes; Fluorescence; Formates; Funding; Galactose Oxidase; Glycine; Goals; Growth and Development function; Histidine; Hormones; Human; Hydrogen Bonding; Investigation; Knowledge; Label; Lead; Libraries; Life; Link; Lyase; Magnetic Resonance Spectroscopy; Maps; Measurement; Measures; Mediating; Medical; Methods; Modeling; Molecular; N2-tryptophyllysine; NMR Spectroscopy; Nature; Organism; Oxidases; Oxidation-Reduction; Oxidative Stress; Pharmacologic Substance; Phenols; Photoreceptors; Physiologic pulse; Plants; Positioning Attribute; Process; Property; Prostaglandin-Endoperoxide Synthase; Proteins; Radial; Reaction; Reporting; Research; Ribonucleotide Reductase; Solutions; Solvents; Spectrum Analysis; Structural Protein; Structure; System; Techniques; Testing; The Sun; Theoretical Studies; Thermodynamics; Tryptophan; Tyrosine; Water; Work; absorption; alpha-glutamyltryptophan; base; carbohydrate metabolism; carcinogenesis; chemical property; chemical reaction; cofactor; density; member; migration; mutant; oxidation; oxidative damage; photosystem II; protein function; public health relevance; quantum; repaired; response; second harmonic; theories; tyrosine analog; 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 amino-acid radical enzymes and pathological conditions linked to dysfunctional amino-acid radical chemistry continuously increases, experimental characterization of parameters involved in protein radical reactions 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 overall aim of the work described here is to use a library of well-structured model proteins specifically made to facilitate electrochemical, structural and spectroscopic studies of tyrosine and tryptophan radicals. During the proposed five-year funding period, we will structurally characterize the reduced and oxidized states of phenol-labeled proteins whose redox properties have already been determined by voltammetry techniques. Solution structures of reduced 2-, 3- and 4-mercaptophenol-13C will be obtained by NMR spectroscopy. The hydrogen-bonding interactions of the oxidized phenols in the 2- and 3-mercaptophenol-13C proteins will be determined by electron magnetic-resonance spectroscopy. The latter project involves cryo-trapping the phenol radicals using a specifically constructed UV/Vis absorption/photolytic spectrometer. The cryo-trapped radicals will be studied by CW and pulsed EPR, ENDOR and ESEEM spectroscopy at 9 and 94 GHz to determine the number and the strength of hydrogen bonds present between the protein matrix and the oxidized phenols. Once the structural studies have been completed, the redox properties of the mercaptophenol-13C proteins will be investigated by quantum chemical methods. Thus, experimental and calculated phenol potentials will be directly connected to protein structural motifs. This level of information is not available for any naturally occurring amino-acid radical. A similar set of voltammetry, structural and theoretical studies is proposed for a model protein enineered to contain a buried tyrosine-histidine hydrogen-bonded complex. Finally, the redox properties of tryptophan will be studied as a function of solvent exposure and electrostatic interactions. The short-term objective with the proposed experimental and theoretical studies is to correlate specific structural features with the reduction potentials and pKa values of tyrosine and tryptophan radicals in the model proteins. The long-term objective with the proposed work is to build a knowledge platform on which to quantitatively model the reduction potentials of functional and dysfunctional amino-acid radicals in natural systems. The studies described in this application have a clear potential to make a significant step towards realizing this long-term objective. PUBLIC HEALTH RELEVANCE: Protein radicals are involved in a range of both beneficial as well as harmful chemical reactions in living organisms. Little is know about these species since they are highly challenging to study experimentally in the natural systems. To increase our knowledge in this research area, we have developed a library of well- structured model proteins specifically made to study the chemical properties of protein radicals.

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状况不断增加，但蛋白质自由基反应中涉及的参数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。这里描述的工作的总体目标是使用一个结构良好的模型蛋白质库，专门用于促进酪氨酸和色氨酸自由基的电化学、结构和光谱研究。在拟议的五年资助期内，我们将从结构上表征酚标记蛋白质的还原态和氧化态，其氧化还原特性已通过伏安技术测定。还原的2-、3-和4-巯基苯酚-13C的溶液结构将通过NMR光谱获得。 2-和3-巯基苯酚-13C 蛋白中氧化酚的氢键相互作用将通过电子磁共振波谱法测定。后一个项目涉及使用专门构造的紫外/可见吸收/光解光谱仪冷冻捕获苯酚自由基。将通过 CW 和脉冲 EPR、ENDOR 和 ESEEM 光谱在 9 和 94 GHz 下研究冷冻捕获的自由基，以确定蛋白质基质和氧化酚之间存在的氢键的数量和强度。一旦结构研究完成，巯基酚-13C 蛋白的氧化还原特性将通过量子化学方法进行研究。因此，实验和计算的苯酚电势将直接与蛋白质结构基序相关。对于任何天然存在的氨基酸残基来说，这一水平的信息是不可用的。针对包含埋藏酪氨酸-组氨酸氢键复合物的模型蛋白质提出了一套类似的伏安法、结构和理论研究。最后，将研究色氨酸的氧化还原性质作为溶剂暴露和静电相互作用的函数。所提出的实验和理论研究的短期目标是将特定结构特征与模型蛋白质中酪氨酸和色氨酸自由基的还原电位和 pKa 值相关联。拟议工作的长期目标是建立一个知识平台，在该平台上对自然系统中功能性和功能失调的氨基酸自由基的还原潜力进行定量建模。本申请中描述的研究显然有可能朝着实现这一长期目标迈出重要一步。公共健康相关性：蛋白质自由基参与生物体中一系列有益和有害的化学反应。人们对这些物种知之甚少，因为在自然系统中对它们进行实验研究非常具有挑战性。为了增加我们在这一研究领域的知识，我们开发了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的化学性质。