Journal
JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY
Volume 52, Issue 1, Pages 219-227Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.yjmcc.2011.09.019
Keywords
Single molecule biophysics; Laser trap; PKA phosphorylation; Contractile proteins; Contractility; Heart
Categories
Funding
- National Institutes of Health [HL086728, HL059408, HL007944, HL07647]
- American Heart Association [0830311N]
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Cardiac myosin binding protein-C (cMyBP-C) has 11 immunoglobulin or fibronectin-like domains, CO through C10. which bind sarcomeric proteins, including titin, myosin and actin. Using bacterial expressed mouse N-terminal fragments (C0 through C3) in an in vitro motility assay of myosin-generated actin movement and the laser trap assay to assess single molecule actin-binding capacity, we determined that the first N-terminal 17 amino acids of the cMyBP-C motif (the linker between C1 and C2) contain a strong, stereospecific actin-binding site that depends on positive charge due to a cluster of arginines. Phosphorylation of 4 serines within the motif decreases the fragments' actin-binding capacity and actomyosin inhibition. Using the laser trap assay, we observed individual cMyBP-C fragments transiently binding to a single actin filament with both short (similar to 20 ms) and long (similar to 300 ms) attached lifetimes, similar to that of a known actin-binding protein, a-actinin. These experiments suggest that cMyBP-C N-terminal domains containing the cMyBP-C motif tether actin filaments and provide one mechanism by which cMyBP-C modulates actomyosin motion generation, i.e. by imposing an effective viscous load within the sarcomere. (C) 2011 Elsevier Ltd. All rights reserved.
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