Structural Framework for Understanding Myosin Thick-Filament Cardiomyopathies

点击翻译按钮获取中文摘要

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.