Unique single molecule binding of cardiac myosin binding protein-C to actin and phosphorylation-dependent inhibition of actomyosin motility requires 17 amino acids of the motif domain

► Actin-binding properties of cardiac myosin binding protein-C (cMyBP-C) defined. ► Single molecule binding of cMyBP-C N-terminal fragments to actin in laser trap. ► First 17 amino acids of the MyBP-C motif define strong actin binding site. ► Phosphorylation of 4 serines within the MyBP-C motif reduces actin binding. ► cMyBP-C may act as a viscous load in the myocardium.