Chemo-Mechanical Energy Transduction

本研究探讨了肌动蛋白和肌球蛋白S1之间的界面在肌肉收缩过程中导致能量的化学机械转导。因此，定点诱变用于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？如果这种亲和差异不是由电荷引起的，而是由肌球蛋白的构象变化引起的，有没有可能只有核磁共振方法才能解释这种构象变化？与肌动蛋白相互作用如何影响“MgADP”中间体向“无配体”中间体的过渡速率？利用位点导向突变和能量最小化模拟来阐明强“无配体”亲和力的结构基础。模拟了具有合适属性的“MgADP”中间体的结构。Shriver/Sykes的核磁共振化学位移发现（在高温度下有一个，在低温度下有两个共存的肌球蛋白构象，通过31-P和19-F探针检测）将指导对各种肌球蛋白分子内交联的研究，这些交联可以捕获这些“MgADP”中间构象，从而确定共存是否包括当前肌球蛋白晶体学“遗漏”的结构。此外，为了了解肌动蛋白对肌球蛋白atp酶的激活作用，研究人员试图干扰那些似乎影响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，以及一个推定的EDC交联位点，包括D471；在C707的大体积组分（如IATR）的激活作用是能量最小化的。共存的构象，如“ATP”和“ADP”之间的转换。Pi”也被检查。这项研究的意义可能在于它从分子上定义了在一个重要的生物收缩系统中使用催化原理进行能量转导。这项研究的目的是了解允许生物实体像机器一样工作的特征和机制-摄取燃料，将其中一些转化为有用的工作，并将其他作为废物产生，而机器本身在这个操作循环中没有遭受净变化。这里正在研究的范例是用于收缩肌肉的生物机器，特别是肌球蛋白蛋白的参与，它吸收燃料并排出废物。用核磁共振等物理方法对肌球蛋白的结构状态和柔韧性的原子细节的静态图片进行了检查，比较了人工改变和自然野生版本。分析了这些变化对运行的影响。此外，还研究了另一种蛋白质肌动蛋白在每一步中对机器运动的锚定作用。尽管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。

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.