Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization.
The structural change that generates force and motion in actomyosin motility has been proposed to be tilting of the myosin light chain domain, which serves as a lever arm. Several experimental approaches have provided support for the lever arm hypothesis; however, the extent and timing of tilting motions are not well defined in the motor protein complex of functioning actomyosin. Here we report three-dimensional measurements of the structural dynamics of the light chain domain of brain myosin V using a single-molecule fluorescence polarization technique that determines the orientation of individual protein domains with 20-40-ms time resolution. Single fluorescent calmodulin light chains tilted back and forth between two well-defined angles as the myosin molecule processively translocated along actin. The results provide evidence for lever arm rotation of the calmodulin-binding domain in myosin V, and support a 'hand-over-hand' mechanism for the translocation of double-headed myosin V molecules along actin filaments. The technique is applicable to the study of real-time structural changes in other biological systems.
Forkey
Joseph N
JN
Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6083, USA.
Quinlan
Margot E
ME
Shaw
M Alexander
MA
Corrie
John E T
JE
Goldman
Yale E
YE
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain; D002147:Calmodulin / Q000378:metabolism; D002642:Chick Embryo; D005454:Fluorescence Polarization; D007700:Kinetics; D009068:Movement; D024701:Myosin Type V / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits
D000199:Actins; D002147:Calmodulin; D000255:Adenosine Triphosphate; D024701:Myosin Type V