A Kinetic Step Involving Loop 1 in Smooth Muscle may Dictate Isoform Specific Differences in ADP Release

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The actin sliding velocity and ADP release rates in smooth muscle myosin are modulated by the make-up of a flexible surface loop spanning the active site known as loop 1. There are only two motor domain isoforms of smooth muscle myosin and they differ in the presence (SM-B) or absence (SM-A) of a seven amino acid insert (QGPSFSY) in this loop. The presence of this insert leads to a two-fold increase in both actin sliding velocity and ADP release, although the mechanism for this difference is unknown. To investigate the role of this insert in functional differences between the SM-B and SM-A isoforms of smooth muscle myosin we have inserted a single tryptophan residue into loop 1 of both isoforms. The dynamics of loop 1 have been correlated with the kinetics of ADP release using a combination of steady-state fluorescence measurements (i.e., tryptophan emission, FRET, and acrylamide quenching) and stopped-flow kinetics. Using this approach we have already shown that the long loop SM-B isoform displays an extra step in its ADP release pathway that has not been previously observed. Here, we show that the additional transition seen in the long loop SM-B isoform is not observed in the short loop SM-A isoform upon ADP release. Furthermore, the final ADP release step is twice as slow in the short loop SM-A isoform, suggesting that the unique transition observed in the presence of the insert alters loop 1 dynamics in a way as to facilitate ADP release. This alteration of ADP release constitutes a simple and fundamental way to tune the activity of the motor at the molecular level and mechanical function at the physiological level in smooth muscle.