Single Myosin Cross-Bridge Orientation in Cardiac Papillary Muscle Detects Lever-Arm Shear Strain in Transduction
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- 作者：Thomas P. Burghardt ; Matthew P. Josephson ; Katalin Ajtai
- 刊名：Biochemistry
- 出版年：2011
- 出版时间：September 13, 2011
- 年：2011
- 卷：50
- 期：36
- 页码：7809-7821
- 全文大小：556K
- 年卷期：v.50,no.36(September 13, 2011)
- ISSN：1520-4995
文摘
Myosin motors transduce ATP free energy into mechanical work. Transduction models allocate specific functions to motor structural domains beginning with ATP hydrolysis in the active site and ending in a lever-arm rotating power-stroke. Myosin light chains, regulatory (RLC) and essential (ELC), bind IQ-domains on the lever-arm and track its movement. Strong evidence exists that light chains stabilize the lever-arm and that light chain mutation undermines stability. Human ventricular RLC tagged with photoactivatable GFP (HCRLC-PAGFP) replaces native RLC in porcine papillary muscle fibers, restores native contractility, and situates PAGFP for single molecule orientation tracking within the crowded fiber lattice. The spatial emission pattern from single photoactivated PAGFP tagged myosins was observed in z-stacks fitted simultaneously to maximize accuracy in estimated dipole orientation. Emitter dipole polar and azimuthal angle pair scatter plots identified an area where steric and molecular crowding constraints depopulated orientations unfavorable for actin interaction. Transitions between pre- and post-power-stroke states represent the lever-arm trajectory sampled by the data and quantify lever-arm shear strain in transduction at three tension levels. These data identify forces acting on myosin in the in situ fiber system due to crowding, steric hindrance, and actomyosin interaction. They induce lever-arm shear strain observed with single molecule orientation detection. A single myosin work histogram reveals discretized power-stroke substates reminiscent of the Huxley鈥揝immons model for myosin based contraction [Huxley and Simmons (1971) Nature233, 533]. RLC or ELC mutation, should it impact lever-arm shear strain, will be detected as changes in single myosin shear strain or power-stroke substate distribution.