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.

Public Health Relevance

Muscle contraction in all healthy cardiac muscle depends not only on functional molecular motors but also on a sound structural framework that allows for the transmission of force. This proposal is directed towards understanding the protein- protein interactions between the coiled-coil regions of the myosin molecule that form the backbone of the thick filament. This fundamental knowledge will be utilized to explain the causes of skeletal and cardiac myopathies that arise from mutations in the myosin rod.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1-MSFC-U (02))
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Adhikari, Bishow B
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University of Wisconsin Madison
Schools of Earth Sciences/Natur
United States
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Andreas, Michael P; Ajay, Gautam; Gellings, Jaclyn A et al. (2017) Design considerations in coiled-coil fusion constructs for the structural determination of a problematic region of the human cardiac myosin rod. J Struct Biol 200:219-228
Korkmaz, E Nihal; Taylor, Keenan C; Andreas, Michael P et al. (2016) A composite approach towards a complete model of the myosin rod. Proteins 84:172-189
Taylor, Keenan C; Buvoli, Massimo; Korkmaz, Elif Nihal et al. (2015) Skip residues modulate the structural properties of the myosin rod and guide thick filament assembly. Proc Natl Acad Sci U S A 112:E3806-15