Structural Framework for Understanding Myosin Thick-Filament Cardiomyopathies

描述（由申请人提供）：健康肌肉中的肌肉收缩不仅取决于功能性分子马达，还取决于允许力传递的健全结构框架。在骨骼肌和心肌中，这种结构是由肌动蛋白附着在相对的Z线上，以及由肌球蛋白卷曲螺旋区组装的双极粗丝提供的。粗肌丝是一个紧密的集合体，肌球蛋白头呈规则的螺旋状排列。这意味着有一个潜在的结构组织。肌球蛋白含有形成这种双极肌丝所必需的信息，然而，即使经过五十多年的研究， 对目前仍不清楚.在初级序列水平和粗肌丝组织的超微结构水平上，关于肌球蛋白杆有丰富的知识。相比之下，肌球蛋白杆的高分辨率结构数据很少。从而 一直不可能产生一个分子模型的粗丝。缺乏知识的原因是肌球蛋白棒的分离片段形成不适合高分辨率结构研究或分子表征的次晶。这个问题现在已经解决，通过纳入适当的增溶结构域，这使得结构的装配能力域的C-末端区域的人？心肌肌球蛋白由X射线晶体学测定。肌球蛋白分子的这一部分对于双极肌丝的形成是必不可少的。本建议的目的是确定这种方法是否可以应用于产生一个高分辨率的整个肌球蛋白棒的结构模型，以及该模型是否可以用来研究肌球蛋白分子之间的相互作用在粗丝。第一个具体目标是确定已被证明影响组装的肌球蛋白棒部分的高分辨率结构，并确定负责形成双极丝的分子特征。作为本研究的一部分，将测量肌球蛋白杆远端节段之间的分子相互作用。这些生物物理学测量将与结构一起建立创建粗丝模型所需的基本分子信息。目前还没有令人满意的生物化学解释的有害影响的心脏和骨骼肌病变的突变位于肌球蛋白杆。这是由于缺乏用于粗灯丝的稳健模型。本建议的第二个具体目的是利用这里确定的片段的结构，以启动结构和生物物理研究，旨在为这些遗传病变的计算模型的背景下，为粗丝提供分子解释。从本研究中获得的见解将适用于所有肌球蛋白II。因此，长期的目标是扩展这里开发的协议，研究平滑肌肌球蛋白丝的结构和组装。

DESCRIPTION (provided by applicant): Muscle contraction in healthy muscle depends not only on a functional molecular motor but also on a sound structural framework that allows for the transmission of force. In skeletal and cardiac muscle this framework is provided by the attachment of actin to the opposing Z-lines and by the bipolar thick filament assembled primarily from the coiled-coil region of myosin. The thick filament is a compact assembly that shows a regular helical disposition of myosin heads. This implies that there is an underlying structural organization. Myosin contains the information necessary to form this bipolar filament, however, even after more than fifty years of investigation, the molecular organization of the thick filament is still unclear. There is a wealth of knowledge concerning the myosin rod at the level of primary sequence and also at the ultrastructural level for the organization of the thick filament. In contrast, there is very little high resolution structural data for the myosin rod. Consequently, it has been impossible to generate a molecular model for the thick filament. The reason for the lack of knowledge is that isolated fragments of the myosin rod form paracrystals that are unsuitable for high resolution structural studies or molecular characterization. This problem has now been solved through the incorporation of appropriate solubilization domains, which has allowed the structure of the Assembly Competence Domain from the C-terminal region of human ??cardiac myosin to be determined by X- ray crystallography. This section of the myosin molecule is essential for bipolar filament formation. The purpose of this proposal is to determine whether this approach can be applied to yield a high resolution structural model for the entire myosin rod and whether this model can be utilized to investigate the interactions between myosin molecules in the thick filament. The first specific aim is to determine the high resolution structure for sections of the myosin rod that have been shown to influence assembly and to determine the molecular features responsible for the formation of bipolar filaments. As part of this study the molecular interactions between distal segments of the myosin rod will be measured. Together with the structures these biophysical measurements will establish the fundamental molecular information necessary to create a model for the thick filament. At present there is no satisfactory biochemical explanation for the deleterious effect of the cardiac and skeletal myopathy mutations located in the myosin rod. This is due to the lack of a robust model for the thick filament. The second specific aim of this proposal is to utilize the structure f fragments determined here to initiate structural and biophysical studies directed towards providing a molecular explanation for these genetic lesions within the context of a computational model for the thick filament. The insight gained from this study will be applicable to all myosin IIs. Thus, the long term goal is to extend the protocols developed here to investigate the structure and assembly of smooth muscle myosin filaments.