Myosin-based kinetic differences between muscle types

描述（由申请人提供）：横纹肌的一个独特特征是其纤维类型的巨大多样性。很大程度上的功能多样性是由于一系列肌球蛋白同种异构体帮助决定肌肉速度。序列比较和分子水平上的功能研究表明，肌球蛋白重链的特定结构域调节过桥循环的动力学步骤，可能影响肌肉速度。我们将利用果蝇系统的独特特征来研究肌球蛋白头部结构和肌肉动力学的异构体差异之间的关系。一项重大进展将是使用肌原纤维制剂，可以精确操纵肌节长度和离子浓度（钙、MgATP、MgADP和磷酸盐），从中我们可以高精度地推断肌动蛋白-肌球蛋白过桥循环的动力学常数。该研究解决了3个基本问题：(i)过桥循环的哪个步骤建立了非常快的肌肉和非常慢的肌肉之间的主要动力学差异？（ii）具有中等速度的光纤类型是否具有介于两个极端之间的过桥速率常数？（iii）肌凝蛋白跨桥循环的哪些步骤受到肌凝蛋白异构体之间特定可变区域变化的影响？为了回答这些问题（作为假设），我们将对比从间接飞行肌（IFM，一种极快的肌肉）、跳跃肌（一种快速肌肉）、从胚胎体壁肌（EMB，一种非常慢的肌肉）转基因表达肌球蛋白的IFM肌原纤维和表达IFM和EMB肌球蛋白嵌合体的IFM肌原纤维中获得的动力学方案。通过将动力学差异与特定结构区域（包括转换区）相关联，我们将根据肌球蛋白的最新结构模型推断分子机制。这项研究的主要优势在于利用果蝇实现了完全整合的方法（从单分子到整个动物），以及使用肌原纤维弥补了分离肌球蛋白实验和剥皮纤维实验之间的差距。

DESCRIPTION (provided by applicant): A unique feature of striated muscle is its enormous diversity of fiber types. Much of the functional diversity is due to a range of myosin isoforms that help determine muscle speed. Sequence comparisons and functional studies at the molecular level have suggested specific structural domains of the myosin heavy chain modulate kinetic steps of the crossbridge cycle that presumably affect muscle speed. We will exploit unique features of the Drosophila system for investigating relationships between isoform differences in myosin head structure and muscle kinetics. A major advance will be the use of myofibrillar preparations that allow precise manipulation of sarcomere length and ion concentrations (calcium, MgATP, MgADP and phosphate), from which we can deduce kinetic constants of the acto-myosin cross-bridge cycle with high precision. The research addresses 3 basic questions: (i) what step(s) of the crossbridge cycle establishes the major kinetic differences between a very fast muscle and a very slow muscle? (ii) do fiber types with intermediate speeds have crossbridge rate constants between those of the extremes? and (iii) what steps of the myosin crossbridge cycle are affected by changes in specific variable region(s) between myosin isoforms? To answer these questions (framed as hypotheses), we will contrast kinetic schemes obtained from myofibrils of indirect flight muscle (IFM, an extremely fast muscle), jump muscle (a fast muscle), IFM myofibrils transgenically expressing myosin from embryonic body wall muscle (EMB, a very slow muscle), and IFM myofibrils expressing chimeras of IFM and EMB myosin. By correlating kinetic differences to specific structural regions (including the converter region), we will deduce molecular mechanisms based on the latest structural models of myosin. The major strengths of this investigation are the fully integrated approach (from single molecules to whole animal) made possible by employing Drosophila, and the use of myofibrils which bridges a gap between experiments with isolated myosin and skinned fiber experiments.