Mutation of a conserved glycine in the SH1-SH2 helix affects the load-dependent kinetics of myosin.
The ATP hydrolysis rate and shortening velocity of muscle are load-dependent. At the molecular level, myosin generates force and motion by coupling ATP hydrolysis to lever arm rotation. When a laser trap was used to apply load to single heads of expressed smooth muscle myosin (S1), the ADP release kinetics accelerated with an assistive load and slowed with a resistive load; however, ATP binding was mostly unaffected. To investigate how load is communicated within the motor, a glycine located at the putative fulcrum of the lever arm was mutated to valine (G709V). In the absence of load, stopped-flow and laser trap studies showed that the mutation significantly slowed the rates of ADP release and ATP binding, accounting for the approximately 270-fold decrease in actin sliding velocity. The load dependence of the mutant's ADP release rate was the same as that of wild-type S1 (WT) despite the slower rate. In contrast, load accelerated ATP binding by approximately 20-fold, irrespective of loading direction. Imparting mechanical energy to the mutant motor partially reversed the slowed ATP binding by overcoming the elevated activation energy barrier. These results imply that conformational changes near the conserved G709 are critical for the transmission of mechanochemical information between myosin's active site and lever arm.
Kad
Neil M
NM
Department of Molecular Physiology & Biophysics, University of Vermont, Burlington, Vermont 05405, USA.
Patlak
Joseph B
JB
Fagnant
Patricia M
PM
Trybus
Kathleen M
KM
Warshaw
David M
DM
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D005998:Glycine / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009119:Muscle Contraction / Q000502:physiology*; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D052898:Optical Tweezers; D011994:Recombinant Proteins; D014633:Valine / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains / Q000502:physiology
D000199:Actins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D018995:Myosin Heavy Chains; D014633:Valine; D005998:Glycine