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: 7104416
• 负责人: PETER LESLIE DUTTON
• 金额: $ 12.8万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7104416
• 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.

在生物能量转换、酶催化、识别和信号传导以及基因作用和调控中，电荷物种的产生、运动和中和是普遍存在的。在所有这些反应的机械根源是定向电场，其跨越长度尺度从催化位点的几埃到生物能量膜上的几十埃。测量和理解生物学中电场的产生、传播和抑制是一个主要的无知领域，也是一个技术挑战。我们通过从头设计的合成蛋白质模型的独特优势，迎接自然工程师和利用电场的挑战， 结构化，高度简化的版本的自然对应物。使用有机化学和分子生物学的组合，maquettes已被设计为具有实验灵活性，以专门解决如何从氧化还原辅因子氧化和还原过程中蛋白质内部产生的电荷发出的电场可以通过蛋白质传播。我们的目标是了解如何在蛋白质领域可以控制的方向和强度，以调节原位氧化还原中心和结合底物的性质，从而管理他们的呼吸电子转移，质子交换和易位和酶催化的活动。两个家庭的maquettes，纳入了一系列的关键机械元素的生物氧化过程的功能代表将被使用。一个是二血红素4- α-螺旋蛋白，另一种是3-α-螺旋蛋白，包括自由基形成侧链酪氨酸和色氨酸或底物醌和烟酰胺。内部和外部电场将产生和测量电化学和可见光，红外和NMR光谱，利用战略性地放置在整个蛋白质的光谱（斯塔克）探针。大规模的领域将被监测的电致变色位移的maquettes含有多烯衍生物的视网膜和胡萝卜素和叶绿素分子。我们的目标是了解蛋白质电场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，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。利用我们的见解设计新蛋白质为我们的新理解提供了最严格的实验测试。