Influence of lever structure on myosin 5a walking.
Using electron microscopy and image processing, we have observed myosin 5a modified with lever arms of different lengths (four, six, and eight calmodulin-binding IQ domains) and orientations walking along actin filaments. Step lengths were dependent on lever length: 8IQ > 6IQ > 4IQ, which is consistent with myosin 5a having evolved to walk straight along actin. Lead heads were mostly in the prepowerstroke state, tethered there by the trail head. However, improved image processing showed that in 5-10% of molecules the lead motor was in the postpowerstroke state. This is a unique attached state of myosin, where the motor domain has completed its powerstroke at the expense of severe lever distortion, but with little cargo movement. Postpowerstroke lead heads were seen in both wild-type and modified lever molecules, mostly where there was least strain. These data allow the strain dependence of the equilibrium between pre- and postpowerstroke conformations to be measured. Slow rates of ADP dissociation observed from lead heads of these molecules can be explained by the unfavorable equilibrium between the pre- and postpowerstroke conformations preceding ADP loss.
Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
Olusola A
OA
Oke
Stan A
SA
Burgess
Eva
E
Forgacs
Peter J
PJ
Knight
Takeshi
T
Sakamoto
James R
JR
Sellers
Howard
H
White
John
J
Trinick
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods*; D008958:Models, Molecular; D009154:Mutation; D024701:Myosin Type V / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D011485:Protein Binding / Q000187:drug effects; D018411:Spodoptera
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D024701:Myosin Type V; D005033:Ethylmaleimide