Structural Framework for Understanding Myosin Thick-Filament Cardiomyopathies

了解肌球蛋白粗丝心肌病的结构框架

• 批准号: 8606771
• 负责人: IVAN RAYMENT
• 金额: $ 20.94万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8606771
• 关键词: Accounting; Actins; Address; Applications Grants; Binding; Biochemical; Biological; C-terminal; Cardiac; Cardiac Myosins; Cardiomyopathies; Competence; Computer Simulation; Data; Development; Distal; Distant; Experimental Models; Filament; Genetic; Goals; Head; Health; Human; Investigation; Ionic Strengths; Knowledge; Laboratories; Lead; Lesion; Location; Measures; Methodology; Modeling; Molecular; Molecular Models; Molecular Motors; Muscle; Muscle Contraction; Mutation; Myocardium; Myopathy; Myosin ATPase; Myosin Rod; Proteins; Protocols documentation; Resolution; Sequence Analysis; Side; Skeletal Muscle; Smooth Muscle; Smooth Muscle Myosins; Structural Models; Structure; Thick Filament; Time; Vertebral column; Vision; Work; X-Ray Crystallography; base; beta-Myosin; biophysical properties; insight; member; molecular assembly/self assembly; molecular modeling; multidisciplinary; non-muscle myosin; protein protein interaction; retinal rods; skeletal; sound; transmission process

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.

描述（由申请人提供）：健康肌肉的肌肉收缩不仅取决于功能性分子马达，还取决于允许传递力的健全的结构框架。在骨骼肌和心肌中，这个框架是由肌动蛋白与相对的 Z 线的附着以及主要由肌球蛋白的卷曲螺旋区域组装而成的双极粗丝提供的。粗丝是一个紧凑的组件，显示出肌球蛋白头的规则螺旋排列。这意味着存在一个底层的结构组织。肌球蛋白包含形成这种双极丝所需的信息，然而，即使经过五十多年的研究，粗丝的分子组织 目前还不清楚。关于肌球蛋白杆在初级序列水平以及粗丝组织的超微结构水平上有丰富的知识。相比之下，肌球蛋白杆的高分辨率结构数据非常少。因此，它 不可能生成粗丝的分子模型。缺乏知识的原因是肌球蛋白杆的孤立片段形成副晶体，不适合高分辨率结构研究或分子表征。这个问题现在已经通过加入适当的增溶结构域得到了解决，这使得可以通过X射线晶体学来确定来自人心肌肌球蛋白C末端区域的组装能力结构域的结构。肌球蛋白分子的这一部分对于双极丝的形成至关重要。该提案的目的是确定这种方法是否可以应用于产生整个肌球蛋白杆的高分辨率结构模型，以及该模型是否可以用于研究粗丝中肌球蛋白分子之间的相互作用。第一个具体目标是确定肌球蛋白杆部分的高分辨率结构，这些部分已被证明会影响组装，并确定负责双极丝形成的分子特征。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。这些生物物理测量结果与结构一起将建立创建粗丝模型所需的基本分子信息。目前，对于位于肌球蛋白杆中的心脏和骨骼肌病突变的有害影响，还没有令人满意的生化解释。这是由于缺乏粗丝的稳健模型。该提案的第二个具体目标是利用此处确定的结构 f 片段来启动结构和生物物理研究，旨在在粗丝计算模型的背景下为这些遗传损伤提供分子解释。从这项研究中获得的见解将适用于所有肌球蛋白 II。因此，长期目标是扩展此处开发的协议以研究平滑肌肌球蛋白丝的结构和组装。