Understanding protein radicals

了解蛋白质自由基

• 批准号: 8268508
• 负责人: Cecilia Tommos
• 金额: $ 22.38万
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8268508
• 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 Repair; DNA biosynthesis; Development; Drug Metabolic Detoxication; Electron Nuclear Double Resonance; Electrons; Electrostatics; Engineering; Environment; Enzymes; Fluorescence Spectroscopy; Formates; Funding; Galactose Oxidase; Glycine; Goals; Growth and Development function; Health; Histidine; Hormones; Human; Hydrogen Bonding; Investigation; Knowledge; Label; Lead; Libraries; Life; Light; 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; S-Adenosylmethionine; Solutions; Solvents; Spectrum Analysis; Structural Protein; Structure; System; Techniques; Testing; 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; quantum; research study; 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了生物体中的一系列生产和破坏性过程。功能性且通常受到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。相比之下，众所周知，氧化应激条件会产生一系列不受控制的范围，对于生物体，可能是有害的蛋白质自由基反应。尽管事实是，与功能失调的氨基酸自由基化学相关的氨基酸自由基酶的数量和病理状况不断增加，但与蛋白质自由基反应有关的参数的实验表征仍然是基本的。这种情况反映了一个简单的事实，即具有反应性和热力学“热”激进状态在自然系统中研究高度挑战。这里描述的工作的总体目的是使用专门制作的结构良好模型蛋白的库来促进酪氨酸和色氨酸自由基的电化学，结构和光谱研究。在拟议的五年资金期间，我们将在结构上表征苯酚标记蛋白的降低和氧化状态，其氧化还原特性已经通过伏安法技术确定。 NMR光谱法将获得降低2-，3和4-甲基苯酚13C的溶液结构。氧化苯酚在2-甲基苯酚-13C蛋白中的氢键相互作用将由电子磁共声光谱确定。后一个项目涉及使用特定构建的UV/VIS吸收/光解光谱仪使用冷冻诱捕苯酚自由基。 CW和脉冲EPR，Endor和ESEEM光谱在9和94 GHz时将研究冷冻捕获的自由基，以确定蛋白质基质和氧化酚类之间存在的氢键的数量和强度。结构研究完成后，将通过量子化学方法研究近载烯醇-13C蛋白的氧化还原特性。因此，实验和计算的酚电位将直接连接到蛋白质结构基序。任何天然发生的氨基酸自由基都无法使用此水平。为模型蛋白含有类似的伏安法，结构和理论研究，该模型含有包含埋藏的酪氨酸 - 硫醇氢键复合物。最后，将研究色氨酸的氧化还原特性作为溶剂暴露和静电相互作用的函数。与拟议的实验和理论研究的短期目标是将特定的结构特征与模型蛋白中酪氨酸和色氨酸自由基的降低电位和PKA值相关联。拟议工作的长期目标是建立一个知识平台，以定量对自然系统中功能和功能失调的氨基酸自由基的减少潜力进行定量模拟。本应用程序中描述的研究具有明显的潜力，可以迈出重要的一步，以实现这一长期目标。公共卫生相关性：蛋白质自由基参与活生物体中的一系列有益和有害的化学反应。对这些物种的了解很少，因为它们在自然系统中进行实验研究非常具有挑战性。为了提高我们在该研究领域的知识，我们开发了一个专门研究蛋白质自由基化学性质的结构化模型蛋白质库。