A 7-amino-acid insert in the heavy chain nucleotide binding loop alters the kinetics of smooth muscle myosin in the laser trap

Two smooth muscle myosin heavy chain isoforms differ by a 7-amino-acid inse rt in a flexible surface loop located near the nucleotide binding site. The non-inserted isoform is predominantly found in tonic muscle, while the ins erted isoform is mainly found in phasic muscle. The inserted isoform has tw ice the actin-activated ATPase activity and actin filament velocity in the in vitro motility assay as the non-inserted isoform. We used the laser trap to characterize the molecular mechanics and kinetics of the inserted isofo rm ((+)insert) and of a mutant lacking the insert ((-)insert), analogous to the isoform found in tonic muscle. The constructs were expressed as heavy meromyosin using the baculovirus/insect cell. system. Unitary displacement (d) was similar for both constructs (similar to 10 nm) but the attachment t ime (t(on) for the (-)insert was twice as long as for the (+)insert regardl ess of the [MgATP]. Both the relative average isometric force (Favg(-insert )/Favg(+-insert)) = 1.1 +/- 0 2 (mean +/- SE) using the in vitro motility m ixture assay, and the unitary force (F similar to 1 pN) using the laser tra p, showed no difference between the two constructs. However, as under unloa ded conditions, t(on) under loaded conditions was longer for the (-)insert compared with the (+)insert construct at limiting [MgATP]. These data sugge st that the insert in this surface loop does not affect the mechanics but r ather the kinetics of the cross-bridge cycle. Through comparisons of t(on) from d measurements at various [MgATP], we conclude that the insert affects two specific steps in the cross-bridge cycle, that is, MgADP release and M gATP binding. (C) Kluwer Academic Publishers.