We recently demonstrated that the widely expressed myosin-I isoform, myo1b, is exquisitely sensitive to tension (Laakso et al. 2008. Science. 321:133-6), where it transitions from a low duty-ratio to a high duty-ratio motor at very low opposing forces (< 1 pN). These forces are transmitted to the motor through the IQ-motif-containing light-chain-binding-domain (LCBD), which is structurally stabilized by calmodulin molecules. Calcium binding to these calmodulins affects the ATPase and motile properties of myo1b (Coluccio & Geeves. 1999. J. Biol. Chem. 274:21575-80). Using stopped-flow fluorescence, we confirmed that calcium accelerates the biochemical rates of phosphate and ADP release by 2 -5 fold. We performed single molecule optical-trap experiments in the presence of 25 mM ATP and 0, 1 or 9 mM free calcium. At low forces in the presence of calcium, we found an acceleration of the actin detachment kinetics, which is consistent with stopped-flow measurements. We also found that the average displacement of the myo1b step decreases to ~0 nm. The decoupling of the LCBD displacement from the motor-domain kinetics prompted us to test how calcium impacts the force sensitivity of actin detachment kinetics. Using an isometric clamp, the addition of 9 mM calcium resulted in a 5-fold decrease in the distance parameter that describes force sensitivity. Finally, we measured the kinetics of calcium binding to myo1b and determined that it occurs in two steps. The first step is very fast and calcium dependent, while the second step is significantly slower and independent of calcium concentration. These results show clearly that calcium regulates the ability of myo1b to sense and sustain tension.
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