Chemo-Mechanical Energy Transduction

化学机械能量转换

• 批准号: 9603670
• 负责人: Manuel Morales
• 金额: $ 20万
• 依托单位: University of the Pacific
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-11-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9603670&HistoricalAwards=false
• 关键词: Chemo; Mechanical; Energy; Transduction

Morales 9603670 Technical This study examines the interface between actin and myosin S1 that lead to chemomechanical transduction of energy during muscle contraction. Thus site-directed mutagenesis is used to specify regions responsible for the hydrophobic and coulombic interactions, and to deduce the involvement of myosin ATPase. Specific questions are: 1 Why do the actin affinities for the "no ligand" and the MgATP" intermediates differ by a thousand fold? 2 Is it possible that only NMR methods can explain this conformational change, if this affinity difference is not due to charge, but to a myosin conformational change? and 3 How is the transition rate of the "MgADP" intermediate to the "no ligand" intermediate influenced by interaction with actin? Site-directed mutations and simulations with energy minimizations are used to elucidate the structural basis of the strong "no ligand" affinity. A structure of the "MgADP" intermediate with suitable attributes is simulated. The NMR chemical shift findings of Shriver/Sykes (At high T there is one, and at low T there are two coexisting conformers of myosin, detectable by 31-P and 19-F probes) will be followed to guide studies on a variety of intramolecular myosin crosslinks which trap these "MgADP" intermediary conformers, so that whether coexistence includes structures "missed" by current myosin crystallography can be determined. Furthermore, as an effort to understand actin activation of myosin ATPase, attempts are made to perturb structures that appear to affect the MgADP "leaving rate". The sites of interest are E99 and E100 for actin, K572, K574, D463 and Y135 for myosin, and a putative EDC crosslink site including D471; the activating effect of a bulky constituent (e.g. IATR) at C707 is energy-minimized. "Coexisting conformers such as the transitions between "ATP" and "ADP.Pi" are also examined. The significance of this study may lie in its molecularly defining the use of catalysis principles in energy-transduction in an important biological contratile system. Non-Technical The aim of this research is to understand the features and mechanisms that allow a biological entity to function as a machine - that takes in a fuel , transforms some of which into useful work, and generates others as waste , while the machine itself suffers no net change in this operational cycle. The paradigm being studied here is the biological machine used to contract muscle, particularly the involvement of the protein myosin which absorbs the fuel as well as ejects the waste. Static pictures in atomic detail of the structural states and flexibility of myosin are examined with physical methods such as nuclear magnetic resonance, comparing artificially altered and the natural wild versions. The effect of these alterations on operation is analyzed. Anchorage of the movement of the machine at each step by another protein, actin , is also studied. Although the central purpose of this research is to figure out how the muscle machine works, it is expected that the knowledge so gained will be used by researchers to understand, construct and repair biological machines in general, or to copy the extraordinary attributes of natural machines into man-made entities.

Morales 9603670 技术本研究检查了肌动蛋白和肌球蛋白S1之间的界面，导致肌肉收缩期间能量的化学力学转导。因此，定点突变被用来指定负责疏水和库仑相互作用的区域，并推断肌球蛋白ATP酶的参与。具体的问题是：1为什么肌动蛋白对“无配体”和MgATP”中间体的亲和力相差一千倍？ 2.如果这种亲和力差异不是由于电荷，而是由于肌球蛋白构象变化，那么是否可能只有NMR方法才能解释这种构象变化？和3“MgADP”中间体到“无配体”中间体的转变速率如何受到与肌动蛋白相互作用的影响？定点突变和能量最小化的模拟用于阐明强“无配体”亲和力的结构基础。模拟具有合适属性的“MgADP”中间体的结构。将遵循Shriver/Sykes的NMR化学位移研究结果（在高T下有一个，在低T下有两个共存的肌球蛋白构象，可通过31-P和19-F探针检测），以指导对捕获这些“MgADP”中间构象的各种分子内肌球蛋白交联的研究，从而可以确定共存是否包括当前肌球蛋白晶体学“遗漏”的结构。此外，作为理解肌动蛋白激活肌球蛋白ATP酶的努力，试图扰动似乎影响MgADP“离开率”的结构。感兴趣的位点是肌动蛋白的E99和E100，肌球蛋白的K572、K574、D463和Y135，以及推定的EDC交联位点，包括D471;在C707处的大体积组分（例如IATR）的激活效应是能量最小化的。同时还研究了共存构象，如“ATP”和“ADP.Pi”之间的转换。 这一研究的意义可能在于从分子水平上界定了催化原理在一个重要的生物控制系统中的能量传递中的应用。 这项研究的目的是了解允许生物实体作为机器发挥作用的特征和机制-它吸收燃料，将其中一些转化为有用的工作，并产生其他废物，而机器本身在这个操作周期中没有净变化。 这里研究的范例是用于收缩肌肉的生物机器，特别是蛋白质肌球蛋白的参与，它吸收燃料并排出废物。 肌球蛋白的结构状态和灵活性的原子细节的静态图片检查与物理方法，如核磁共振，比较人工改变和自然野生版本。 分析了这些变化对运行的影响。锚定的运动的机器在每一步的另一种蛋白质，肌动蛋白，也进行了研究。虽然这项研究的中心目的是弄清楚肌肉机器是如何工作的，但预计研究人员将利用由此获得的知识来理解，构建和修复一般的生物机器，或者将自然机器的非凡属性复制到人造实体中。