Year-end Sale % OFF 
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.
Highlights
Globular head with actin filament facilitates the release of ATP hydrolysis products from myosin
Exchange of the essential light chain (ELC) in Permeabilized Muscle Fibers—To investigate the interface region of the lever arm, we developed a protocol to substitute the native ELC with a fluorescent recombinant ELC in isolated skeletal muscle fibers
From the band corresponding to the ELC in the treated muscle fiber, the proportion of the exchanged ELC was found as the difference between the isolated ELC and the treated fiber staining ratios
Summary
Globular head with actin filament facilitates the release of ATP hydrolysis products from myosin. There is still no crystal structure of actomyosin available, x-ray diffraction [4] and fluorescence polarization studies of both RLC [5,6,7] and ELC [8] confirmed rotation of the lever arm when comparing active, relaxed, and rigor muscle fibers. The aim of this work is to investigate the response of crossbridges to the chemo-mechanical transitions in muscle fibers by probing the microenvironment of the interface region of the myosin lever arm domain. Local environmental changes can be detected by other methods, fluorescence polarization, their interpretation is often complicated by other contributing factors, such as changes in orientation and protein mobility For this purpose, we utilized the fluorescence lifetime of coumarin covalently bound to the interface. The results showed a response of the C terminus of the ELC to the stretch, with the amplitude of response depending on location of the probe
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
