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

Lauzon AM, Tyska MJ, Rovner AS, Freyzon Y, Warshaw DM, Trybus KM
J Muscle Res Cell Motil. 1998 19 (8): 825-37

PMID: 10047983 · 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.

MeSH Terms (14)

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

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