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Weith, Abbey

Molecular Physiology and Biophysics

2012

PhD

Cardiac myosin binding protein-C (cMyBP-C) is a sarcomeric protein with 11 domains (C0-C10) that modulates actomyosin interactions in order to regulate cardiac contractility. It accomplishes this through its C-terminal attachment to myosin LMM, which anchors it in the thick filament, and its N-terminal binding to myosin S2 and actin. cMyBP-C's N-tenninus also contains 4 phosphorylation sites within the C1-C2 linker, which presumably attenuate both its myosin and actin binding capacities when phosphorylated. The specificity and importance of cMyBP-C's N-terminal actin binding capacity as well as the ability of phosphorylation to modulate actin binding is still under debate. In these studies we used single molecule biophysical techniques (i.e. in vitro motility and laser trap assays) and bacterially expressed N-tenninal cMyBP-C fragments (varying domains C0 through C3) to investigate the specificity of cMyBP-C's actin binding and define the domains necessary for this interaction. We also examined if phosphorylation regulates cMyBP-C's actin binding and whether partial phosphorylation (singly versus doubly phosphorylated etc.) impacts cMyBP-C function.
Using structural deletions and amino acid substitutions, we identified an actin-binding site within the first 17 amino acids of the C1-C2 linker following the C1 domain that may interact with actin stereospecifically. All N-terminal fragments studied with these 17 amino acids inhibit actomyosin motility to the same extent as whole cMyBP-C. Phosphorylation of the N-terminal fragments reduced their inhibition of actomyosin 3-4 fold in the motility assay, which could be explained by a reduction in actin binding. Based on direct evidence that the N-terminal fragments transiently bind to single actin filaments in the laser trap assay, we propose that cMyBP-C may exert its mechanical effects on actomyosin by creating a physical link between the thick and thin filaments that imposes an internal viscous load within the sarcomere. Phosphorylation of cMyBP-C reduces its ability to bind actin, which could the break the link and relieve the load.
To examine the effects of partial phosphorylation, we expressed eight different C0-C3 constructs with alanines to mimic dephosphorylation or aspartic acids for phosphorylation at 3 sites, 8273, 8282 and 8302. Phosphorylation had a bimodal effect on C0-C3's inhibition of velocity in the motility assay; singly phosphorylated C0-C3 inhibited velocity similarly to unphosphorylated C0-C3, while doubly and triply phosphorylated constructs were less inhibitory to approximately the same extent. In the load clamped laser trap assay, used to examine the C0-C3 constructs' effects on actomyosin's force-velocity (FV) relationship and power generation, several fragments had inhibitory effects on velocities at lower loads that roughly matched their degree of modulation in the motility assay.
This resulted in a depression ofthe FV relationship, which was fit by a modified Hill equation assuming cMyBP-C acts as a viscous load. Viscosity coefficients up to 4.7 pN*([mu]m/s) were obtained for C0-C3 ensembles in various phosphorylation states, while individual C0-C3 molecules had estimated viscosity coefficients up to 0.4 pN*([mu]m/s). Duplicating the bimodal trend from the motility assay, singly phosphorylated C0-C3 reduced peak power ~40% like unphosphorylated C0-C3, while neither doubly or triply phosphorylated constructs altered power. These data also support a model in which cMyBP-C acts as a viscous load through N-terminal interactions with actin, which can be modulated via phosphorylation in an "on/off' manner.