Single-myosin crossbridge interactions with actin filaments regulated by troponin-tropomyosin.
Striated muscle contraction is governed by the thin filament regulatory proteins troponin and tropomyosin. Here, we investigate the molecular mechanisms by which troponin-tropomyosin inhibits myosin's interactions with the thin filament in the absence of calcium by using a laser trap. The displacement events for a single-myosin molecule interacting with a reconstituted thin filament were shorter (step size = 5 nm) and prolonged (69 ms) compared with actin alone (11 nm and 26 ms, respectively). However, these changes alone do not account for the degree of inhibition of thin filament movement observed in an ensemble assay. Our investigations of single- and multiple-myosin molecules with regulated thin filaments suggest the primary basis for this inhibition derives from an approximately 100-fold decrease in the probability of myosin attaching to actin. At higher myosin concentrations, short bursts of motility are observed in a laser trap consistent with the strong binding of a single-myosin crossbridge, resulting in cooperative binding of other cycling crossbridges. We confirmed this cooperativity in the in vitro motility assay by observing thin filament translocation in the absence of calcium but at low [ATP], consistent with rigor activation. We have developed a simple mechanistic model that reproduces and provides insight into both the observed single-myosin molecule and ensemble data in the absence of Ca(2+). These data support the hypothesis that thin filament inhibition in the absence of Ca(2+) is largely achieved by modulating the rate of attachment and/or transition from the weakly to strongly bound state.
Kad
Neil M
NM
Departments of Molecular Physiology and Biophysics and Medicine, University of Vermont, Burlington, VT 05405, USA.
Kim
Scott
S
Warshaw
David M
DM
VanBuren
Peter
P
Baker
Josh E
JE
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000502:physiology; D002645:Chickens; D003198:Computer Simulation; D008274:Magnesium / Q000378:metabolism; D008954:Models, Biological; D009010:Monte Carlo Method; D009119:Muscle Contraction / Q000502:physiology; D018482:Muscle, Skeletal / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D014335:Tropomyosin / Q000737:chemistry* / Q000502:physiology; D014336:Troponin / Q000737:chemistry* / Q000502:physiology
D000199:Actins; D014335:Tropomyosin; D014336:Troponin; C042260:troponin-tropomyosin complex; D000255:Adenosine Triphosphate; D009218:Myosins; D008274:Magnesium; D002118:Calcium