Characterization of three regulatory states of the striated muscle thin filament.
The troponin-tropomyosin-linked regulation of striated muscle contraction occurs through allosteric control by both Ca(2+) and myosin. The thin filament fluctuates between two extreme states: the inactive "off" state and the active "on" state. Intermediate states have been proposed from structural studies and transient kinetic measurements. However, in contrast to the well-characterised, on and off states, the mechanochemical properties of the intermediate states are much less well understood because of the instability of those states. In the present study, we have characterized a myosin-induced intermediate that is stabilized by cross-linking myosin motor domains (S1) to actin filaments (with a maximum of one S1 molecule for 50 actin monomers). A single S1 molecule is known to interact with two adjacent actin monomers. A detailed analysis revealed that thin filaments containing S1 molecules cross-linked to just one actin monomer (actin(1)-S1 complexes) are regulated with a 79% inhibition of the ATPase in the absence of Ca(2+). In contrast, filaments containing S1 molecules cross-linked at two positions, to two adjacent actin monomers (actin(2)-S1 complexes) totally lose their regulation in a highly cooperative manner. This loss of regulation was due both to an enhancement of the ATPase activity without calcium and an inhibition of the ATPase with calcium. Filaments containing actin(2)-S1 complexes, with significant ATPase activity in the absence of calcium (about 50%), did not move on a myosin-coated surface unless calcium was present. This partial uncoupling between the ATPase activity and in vitro motility in the absence of calcium demonstrates that the mechanical steps require actin-myosin contacts, which take place only in the on state and not in the off or intermediate states. These data provide new insights concerning the difference in cooperativity of Ca(2+) regulation that exists between the biochemical and mechanical cycles of the actin-myosin motor.
Van Dijk
Juliette
J
UPR1086-CNRS, IFR24, Montpellier, France.
Knight
Alex E
AE
Molloy
Justin E
JE
Chaussepied
Patrick
P
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D020409:Molecular Motor Proteins / Q000378:metabolism; D009119:Muscle Contraction / Q000502:physiology*; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011817:Rabbits; D014336:Troponin / Q000378:metabolism
D003432:Cross-Linking Reagents; D020409:Molecular Motor Proteins; D020033:Protein Isoforms; D014336:Troponin; D000251:Adenosine Triphosphatases; D009218:Myosins; D005022:Ethyldimethylaminopropyl Carbodiimide; D002118:Calcium