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 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.
Lauzon
A M
AM
Department of Molecular Physiology and Biophysics, University of Vermont, Burlington 05405, USA.
Tyska
M J
MJ
Rovner
A S
AS
Freyzon
Y
Y
Warshaw
D M
DM
Trybus
K M
KM
D000199:Actins / Q000737:chemistry / Q000502:physiology; D000255:Adenosine Triphosphate / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D007700:Kinetics; D007834:Lasers; D020409:Molecular Motor Proteins / Q000502:physiology; D009119:Muscle Contraction / Q000502:physiology; D009130:Muscle, Smooth / Q000737:chemistry* / Q000502:physiology; D018995:Myosin Heavy Chains / Q000737:chemistry* / Q000502:physiology*; D010777:Photochemistry; D017434:Protein Structure, Tertiary; D013314:Stress, Mechanical
D000199:Actins; D020409:Molecular Motor Proteins; D000255:Adenosine Triphosphate; D018995:Myosin Heavy Chains