Response of rigor cross-bridges to stretch detected by fluorescence lifetime imaging microscopy of myosin essential light chain in skeletal muscle fibers |
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Authors: | Ushakov Dmitry S Caorsi Valentina Ibanez-Garcia Delisa Manning Hugh B Konitsiotis Antonios D West Timothy G Dunsby Christopher French Paul M Ferenczi Michael A |
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Institution: | National Heart and Lung Institute, Imperial College London, London SW7 2AZ, United Kingdom. d.ushakov@imperial.ac.uk |
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Abstract: | We applied fluorescence lifetime imaging microscopy to map the microenvironment of the myosin essential light chain (ELC) in permeabilized skeletal muscle fibers. Four ELC mutants containing a single cysteine residue at different positions in the C-terminal half of the protein (ELC-127, ELC-142, ELC-160, and ELC-180) were generated by site-directed mutagenesis, labeled with 7-diethylamino-3-((((2-iodoacetamido)ethyl)amino)carbonyl)coumarin, and introduced into permeabilized rabbit psoas fibers. Binding to the myosin heavy chain was associated with a large conformational change in the ELC. When the fibers were moved from relaxation to rigor, the fluorescence lifetime increased for all label positions. However, when 1% stretch was applied to the rigor fibers, the lifetime decreased for ELC-127 and ELC-180 but did not change for ELC-142 and ELC-160. The differential change of fluorescence lifetime demonstrates the shift in position of the C-terminal domain of ELC with respect to the heavy chain and reveals specific locations in the lever arm region sensitive to the mechanical strain propagating from the actin-binding site to the lever arm. |
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Keywords: | Fluorescence Microscopic Imaging Molecular Motors Myosin Protein Conformation Protein-Protein Interactions Skeletal Muscle Fluorescence Lifetime Imaging Microscopy |
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