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

J Muscle Res Cell Motil. 1998 Nov;19(8):825-37. doi: 10.1023/a:1005489501357.


Two smooth muscle myosin heavy chain isoforms differ by a 7-amino-acid insert in a flexible surface loop located near the nucleotide binding site. The non-inserted isoform is predominantly found in tonic muscle, while the inserted isoform is mainly found in phasic muscle. The inserted isoform has twice 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 isoform ((+)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 (approximately 10 nm) but the attachment time (t(on) for the (-)insert was twice as long as for the (+)insert regardless of the [MgATP]. Both the relative average isometric force (Favg(-insert)/Favg(+insert) = 1.1 +/- 0.2 (mean +/- SE) using the in vitro motility mixture assay, and the unitary force (F approximately 1 pN) using the laser trap, showed no difference between the two constructs. However, as under unloaded conditions, t(on) under loaded conditions was longer for the (-)insert compared with the (+)insert construct at limiting [MgATP]. These data suggest that the insert in this surface loop does not affect the mechanics but rather the kinetics of the cross-bridge cycle. Through comparisons of t(on) from d measurements to various [MgATP], we conclude that the insert affects two specific steps in the cross-bridge cycle, that is, MgADP release and MgATP binding.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / chemistry
  • Actins / physiology
  • Adenosine Triphosphate / physiology
  • Amino Acid Sequence
  • Animals
  • Chickens
  • Kinetics
  • Lasers*
  • Molecular Motor Proteins / physiology
  • Muscle Contraction / physiology
  • Muscle, Smooth / chemistry*
  • Muscle, Smooth / physiology
  • Myosin Heavy Chains / chemistry*
  • Myosin Heavy Chains / physiology*
  • Photochemistry
  • Protein Structure, Tertiary
  • Stress, Mechanical


  • Actins
  • Molecular Motor Proteins
  • Adenosine Triphosphate
  • Myosin Heavy Chains