Step-size is determined by neck length in myosin V.
The highly processive motor, myosin V, has an extremely long neck containing six calmodulin-binding IQ motifs that allows it to take multiple 36 nm steps corresponding to the pseudo-repeat of actin. To further investigate how myosin V moves processively on actin filaments, we altered the length of the neck by adding or deleting IQ motifs in myosin constructs lacking the globular tail domain. These myosin V IQ mutants were fluorescently labeled by exchange of a single Cy3-labeled calmodulin into the neck region of one head. We measured the step-size of these individual IQ mutants with nanometer precision and subsecond resolution using FIONA. The step-size was proportional to neck length for constructs containing 2, 4, 6, and 8 IQ motifs, providing strong support for the swinging lever-arm model of myosin motility. In addition, the kinetics of stepping provided additional support for the hand-over-hand model whereby the two heads alternately assume the leading position. Interestingly, the 8IQ myosin V mutant gave a broad distribution of step-sizes with multiple peaks, suggesting that this mutant has many choices of binding sites on an actin filament. These data demonstrate that the step-size of myosin V is affected by the length of its neck and is not solely determined by the pseudo-repeat of the actin filament.
Sakamoto
Takeshi
T
Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1762, USA.
Yildez
Ahmet
A
Selvin
Paul R
PR
Sellers
James R
JR
D008841:Actin Cytoskeleton / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002232:Carbocyanines / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000737:chemistry; D009154:Mutation; D024701:Myosin Type V / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits
D002147:Calmodulin; D002232:Carbocyanines; D005456:Fluorescent Dyes; C089187:cyanine dye 3; D024701:Myosin Type V