The Natural Engineering of Internal Electric Fields in Redox Proteins at Differen

Differen 氧化还原蛋白内部电场的自然工程

• 批准号: 6706156
• 负责人: PETER LESLIE DUTTON
• 金额: $ 12.43万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6706156
• 关键词: Schiff bases; catalyst; charge transfer complex; chemical stability; chemical structure function; chromophore; cofactor; electric field; electron transport; electronic spectra; infrared spectrometry; intermolecular interaction; ionic strengths; molecular dynamics; nuclear magnetic resonance spectroscopy; protein engineering; protein structure function; synthetic protein

Generation, movement and neutralization of charge species are universal in biological energy conversion and enzyme catalysis, recognition and signaling and gene action and regulation. At the mechanistic roots of all these reactions are directed electric fields that span length scales from the few Angstroms of catalytic sites to the tens of Angstroms across bioenergetic membranes. Measuring and understanding the generation, propagation and suppression of electric fields in biology represents a major area of ignorance and is a technical challenge. We meet the challenge of how nature engineers and harnesses electric fields through the unique advantages of de novo designed synthetic protein models, maquettes, that are robust, structured, and highly simplified versions of natural counterparts. Using a combination of organic chemistry and molecular biology, maquettes have been designed with experimental flexibility to specifically address how electric fields emanating from charges generated in the interior of protein during redox cofactor oxidation and reduction may propagate through protein. The goal is to understand how fields in proteins can be controlled in direction and in intensity to modulate in situ properties of redox centers and bound substrates and thereby govern their activity in respiratory electron transfer, proton exchange and translocation and enzyme catalysis. Two families of maquettes that incorporate a range of key mechanistic elements functionally representative of the biological oxidative processes will be used. One is a di-heme 4- alpha-helix protein and another is a 3-alpha-helix protein including radical forming side-chains tyrosine and tryptophan or substrates quinone and nicotinamide. Internal and external electric fields will be generated and measured electrochemically and by visible, infrared and NMR spectroscopy, exploiting strategically placed spectral (Stark) probes throughout the protein. Large scale fields will be monitored by electrochromic shifts in maquettes containing the polyene derivative of retinal and carotenes and chlorophyll molecules. We aim to understand the fundamental means by which protein electric fields are productively managed and how mismanagement contributes to the failure of electron transfer protein that results in diseases arising from either genetic lesions at birth, stress or the natural result of the aging process. The design of new proteins employing our insights provides the most stringent experimental tests of our new understanding.

电荷物种的产生，运动和中和化在生物能转化和酶催化，识别和信号传导以及基因作用和调节中是普遍的。在所有这些反应的机械根上，都是定向电场，这些电场从催化位点的几个埃埃斯特罗姆到跨生物能膜的数十只埃埃斯特罗姆。衡量和了解生物学中电场的产生，传播和抑制是无知的主要领域，并且是技术挑战。我们遇到了自然工程师和利用电场如何通过从头设计的合成蛋白质模型的独特优势的挑战，这些模型是强大的， 自然对应物的结构化和高度简化的版本。使用有机化学和分子生物学的组合，已经设计了具有实验灵活性，以专门解决在氧化还原辅助氧化和还原过程中蛋白质内部产生的电场如何通过蛋白质传播的电场。目的是了解如何以方向和强度控制蛋白质中的领域，以调节氧化还原中心和结合底物的原位特性，从而控制其在呼吸电子转移，质子交换，易位以及酶催化中的活性。将使用两个熟练的毛孔家族，它们将使用一系列在生物氧化过程功能的关键机械元素。一个是二峰4-- α-螺旋蛋白和另一种是3-α-螺旋蛋白，包括自由基形成的侧链酪氨酸和色氨酸或底物喹酮和烟酰胺。内部和外部电场将通过电化学以及可见的，红外和NMR光谱生成和测量，并在整个蛋白质中利用策略性放置的光谱（Stark）探针。大规模田地将通过含有视网膜和胡萝卜素和叶绿素分子的多烯衍生物的斑块中的电染料移位来监测。我们旨在了解蛋白质电场的有效管理的基本手段，以及管理不善如何导致电子转移蛋白的失败，这会导致出生时遗传病变，压力或衰老过程的自然结果引起的疾病。使用我们的见解的新蛋白质的设计为我们的新理解提供了最严格的实验测试。