Actin-Myosin Crossbridge Binding and Regulation Studied Using Force Spectroscopy

Clobes, Amy, Biomedical Engineering - School of Engineering and Applied Science, University of Virginia
Guilford, William, Department of Biomedical Engineering, University of Virginia

Muscle contraction requires a symphony of proteins that act and interact to generate force and movement. Actin and myosin are the essential crossbridge proteins that drive muscle contraction, and they rely on regulatory mechanisms to fine-tune their activity. This regulation can be intramolecular, or it can come from post-translational modifications, or interaction with another protein or small molecule. Because of the myriad of interactions in a sarcomere, it is difficult to discern the presence and significance of each one. Single-molecule and ensemble in vitro models allow us to isolate the effects of a single protein or post-translational modification to a specific component of the contractile apparatus – something that cannot be done in intact fibers or isolated myofibrils. We exploited the capabilities of single-molecule measurements to understand the mechanism of two important regulatory crossbridge interactions.

First, we show that myosin loop 2 internally regulates the crossbridge by acting as a force-dependent inhibitor of the long-lived actomyosin complex. Here, an optical trap was used to measure bond formation and rupture between actin and rigor heavy meromyosin (HMM) with loop 2 intact or tryptically cleaved. When loop 2 was cleaved, actomyosin catch bond behavior was abrogated leaving only a long-lived state – pointing to the integral role of loop 2 and the importance of force in crossbridge regulation.

Cardiac myosin binding protein-C (cMyBP-C) is a sarcomeric protein that directly regulates crossbridge formation and cycling, though its specific binding partner and mechanism of regulation are unknown. Moreover, cMyBP-C activity itself is regulated by phosphorylation and may be regulated by nitrosylation. In order to identify the crossbridge binding partner of cMyBP-C, we measured the force-dependent bond rupture between cMyBP-C and each of the requisite crossbridge components, actin and HMM, individually. We found that neither the bond with actin nor the bond with HMM was regulated by phosphorylation, excluding these as essential crossbridge binding partners for cMyBP-C and disproving the leading hypothesis in the field. Further, our work shows that nitrosylation of cMyBP-C does not play a role in regulating actin-myosin interactions.

These studies have allowed us to gain a better understanding of the protein-protein interactions between actin, myosin, and cMyBP-C that are key mechanisms of crossbridge formation and regulation. Together, our data on the functions of loop 2 and cMyBP-C increase our understanding of striated muscle function and may be especially important in understanding pathologies. Hypertrophic cardiomyopathy in particular is directly linked to mutations in cMyBP-C, and until we have a better understanding of how cMyBP-C regulates crossbridge activity the mechanism of disease will remain unclear.

PHD (Doctor of Philosophy)
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