Probing the reverse directionality of myosin VI using artificial lever arms
Jung-Chi Liao, Zev Bryant, Mary W Elting, Scott L Delp, James A Spudich
Myosin uses ATP chemical energy to perform diverse functions such as muscle contractions, cell division, and vesicle transports. My interests lie in integrating experimental and computational techniques together to understand its mechanics and energy transduction. I have designed myosin VI with artificial lever arms to identify key structural elements of myosin VI dictating its reverse directionality. I implemented computational modeling and molecular dynamics simulation to guide the protein design. I then used in vitro motility experiments, optical tweezers, and total internal reflection fluorescence microscopy to test these designs. Our results demonstrated that the calmodulin-bound portion is not an integral mechanical component of the reverse stroke. Furthermore, we were able to alter the moving direction of myosin on actin filaments with the change of only 18 amino acids. The following movies from in vitro motility experiments show the actin filaments moved by designed myosins toward the opposite directions. Field size: 12.8 μm x 6.4 μm
Two designed myosins moving toward the opposite directions |
||
(-) end directed motor (moving toward the Cy5 red label) |
(+) end directed motor (moving away from the Cy5 red label) |
|
 |
 |
|