Abstract
Myosin II is the main force-generating motor during muscle contraction. Myosin II exists as different isoforms that are involved in diverse physiological functions. One outstanding question is whether the myosin heavy chain (MHC) isoforms alone account for these distinct physiological properties. Unique sets of essential and regulatory light chains (RLCs) are known to assemble with specific MHCs, raising the intriguing possibility that light chains contribute to specialized myosin functions. Here, we asked whether different RLCs contribute to this functional diversification. To this end, we generated chimeric motors by reconstituting the MHC fast isoform (MyHC-IId) and slow isoform (MHC-I) with different light-chain variants. As a result of the RLC swapping, actin filament sliding velocity increased by ∼10-fold for the slow myosin and decreased by >3-fold for the fast myosin. Results from ensemble molecule solution kinetics and single-molecule optical trapping measurements provided in-depth insights into altered chemo-mechanical properties of the myosin motors that affect the sliding speed. Notably, we found that the mechanical output of both slow and fast myosins is sensitive to the RLC isoform. We therefore propose that RLCs are crucial for fine-tuning the myosin function.
Highlights
(i.e. the detection limit with our current experimental setup))
For over 2 decades, the issue of myosin heterogeneity in the muscle fiber has been discussed in the literature, and particular emphasis has been put on the mixed content of myosin isoforms
By reconstituting a homogeneous population of such well-defined hybrid/chimeric motors and analyzing the kinetic and mechanical features of the constructs using single-molecule and ensemble measurements, we were able to unravel the influence of individual RLCs on the mechanical performance of slow- and fast-muscle myosins
Summary
(i.e. the detection limit with our current experimental setup)). The AM-detached state, toff, included the ATP hydrolysi. For slow myosin heavy chain, the S1s-MLC2v and S1s-MLC2B combination resulted in a significant decrease of the average displacement amplitude, with S1sMLC2B displaying merely 1.86-nm stroke size. To investigate this notion of whether the different RLCs influenced the stiffness of the chimeric motors, we acquired the AM interaction records by applying positive feedback on the laser-trapped beads as described by Steffen et al [36, 47].
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