Response of Rigor Cross-bridges to Stretch Detected by Fluorescence Lifetime Imaging Microscopy of Myosin Essential Light Chain in Skeletal Muscle Fibers

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.The function of striated muscles is brought about by the relative sliding of actin and myosin filaments. Muscle myosin is a hexameric protein, assembled from two heavy chains, each bearing a regulatory and an essential light chain (RLC 3 and ELC, respectively). The heavy chains consist of a long tail region responsible for the formation of a filament structure and a globular head containing the catalytic domain and the lever arm, which binds the light chains. The interaction of the globular head with actin filament facilitates the release of ATP hydrolysis products from myosin. The accompanying structural changes result in rotation of the lever arm (1-3).The molecular details of the change in position of the lever arm with respect to the catalytic domain were revealed by crystallographic studies of myosin head in the presence of nucleotide analogues in the ATPase pocket (2). Although there is still no crystal structure of actomyosin available, x-ray diffraction (4) and fluorescence polarization studies of both RLC (5-7) and ELC (8) confirmed rotation of the lever arm when comparing active, relaxed, and rigor muscle fibers. However, the angular changes were substantially different from those expected by crystallography. This discrepancy could be due to the crystal packing or the use of a truncated form of myosin head, which lacked most of the lever arm. In addition, the fluorescence polarization studies were focused on the distal part of the lever arm, which is remote from the catalytic domain. Therefore, the proximal part of the lever arm, which forms an interface with the catalytic domain, is of particular interest. The fluorescence polarization study of this region (9) showed its reorientation when muscle fibers were moved from relaxation to rigor. However, only a single labeling site was used, and the details of this reorientation are still not clear.The aim of this work ...

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