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

• Published in: Journal of muscle research and cell motility 1998-11, Vol.19 (8), p.825-837
• Main Authors: Lauzon, A M, Tyska, M J, Rovner, A S, Freyzon, Y, Warshaw, D M, Trybus, K M
• Format: Article
• Language: English
• Subjects: Actins - chemistry; Actins - physiology; Adenosine Triphosphate - physiology; Amino Acid Sequence; Animals; Binding sites; Chickens; Kinetics; Lasers; Molecular Motor Proteins - physiology; Muscle Contraction - physiology; Muscle, Smooth - chemistry; Muscle, Smooth - physiology; Muscular system; Myosin Heavy Chains - chemistry; Myosin Heavy Chains - physiology; Photochemistry; Protein Structure, Tertiary; Proteins; Stress, Mechanical
• ISSN: 0142-4319; 1573-2657
• 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.