Molecular basis of myosin-2 function
Final Report Abstract
Almost all our knowledge about the mechanochemical interactions of motor proteins is derived from averaged data sets; either from solution studies to determine kinetic parameters or from investigations of the motor activity in a filament gliding assay using an ensemble of motor proteins. Averaging methods are also used in single molecule studies. As a result of averaging procedures a load dependent bias can not be excluded. By following a single molecule of slow non-muscle myosins through the complete actomyosin cross-bridge cycle, we dissect the biochemical and mechanical steps of the cycle from the initial weak binding state to the rigor state. Combining an improved time resolution optical trap with total internal reflection (TIRF) microscopy to assess nucleotide state allowed us to study the coupling of biochemical and molecular events at the single molecule level. The project had a key function in bridging the areas focused on actin and MT-based motility with particular focus on dyneinmediated transport. Our results with actomyosin show that the initial binding of myosin.ADP.Pi to actin is generating a short-lived state with a lifetime well below a millisecond. Using myosins with very slow rates of phosphate release did not extend the lifetime indicating that weak binding is not governed by the rate of phosphate-release. In optical trap experiments two binding states were well-defined: the actomyosin.ADP complex for myosin-5 and actomyosin rigor complex for myosin-2 and -5. While a weak binding state could be initiated by introducing additional positive charges (6 lysines) within loop-2, these additional charges did not appear to extend the pre-power stroke state of myosin.
Publications
- (2010) Cytoplasmic dynein is not a conventional processive motor. J Struct Biol 170: 266-269
Walter WJ, Brenner B, Steffen W
(See online at https://doi.org/10.1016/j.jsb.2009.11.011) - (2010) Plusand minus-end directed microtubule motors bind simultaneously to herpes simplex virus capsids using different inner tegument structures. PLoS Pathog 6: e1000991
Radtke K, Kieneke D, Wolfstein A, Michael K, Steffen W, Scholz T, Karger A, Sodeik B
(See online at https://doi.org/10.1371/journal.ppat.1000991) - (2010) Targeted optimization of a protein nanomachine for operation in biohybrid devices. Angew Chem Int Ed Engl 49: 312-316
Amrute-Nayak M, Diensthuber RP, Steffen W, Kathmann D, Hartmann FK, Fedorov R, Urbanke C, Manstein DJ, Brenner B, Tsiavaliaris G
(See online at https://doi.org/10.1002/ange.200905200) - (2012) Mechanical and kinetic properties of beta-cardiac/slow skeletal muscle myosin. J Muscle Res Cell Motil 33: 403-417
Brenner B, Hahn N, Hanke E, Matinmehr F, Scholz T, Steffen W, Kraft T
(See online at https://doi.org/10.1007/s10974-012-9315-8) - (2012) Two independent switches regulate cytoplasmic dynein's processivity and directionality. Proc Natl Acad Sci USA 109: 5289-5293
Walter WJ, Koonce MP, Brenner B, Steffen W
(See online at https://doi.org/10.1073/pnas.1116315109)
