LaConte Leslie E W, Baker Josh E, Thomas David D
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Biochemistry. 2003 Aug 19;42(32):9797-803. doi: 10.1021/bi034288r.
We have used electron paramagnetic resonance (EPR) of spin-labeled scallop muscle, in conjunction with laser flash photolysis of caged ATP, to resolve millisecond rotational transitions of the myosin light-chain domain (LCD) during transient force generation. We previously used EPR to resolve two distinct orientations of the LCD [Baker, J. E., Brust-Mascher, I., Ramachandran, S., LaConte, L. E., and Thomas, D. D. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2944-2949], correlated these structural states with biochemical states in the actin-myosin ATPase reaction, and showed that a small shift in the steady-state distribution between these two LCD orientations (i.e., a net lever arm rotation) is associated with force generation in muscle. In the study presented here, we measured millisecond changes in this orientational distribution (i.e., the rates of transition between the two LCD orientations) in muscle following flash photolysis of caged ATP, in both the presence and absence of Ca. The transient acquired in the absence of Ca is dominated by a rapid (1/tau(1) = 37 s(-1)) disordering transition from the single orientation in rigor to the bimodal orientation distribution observed for detached cross-bridges in relaxation (i.e., the reversal of the lever arm rotation), followed by a recovery phase (1/tau(2) = 2.4 s(-1)) of very small amplitude (small fraction of heads participating). In the presence of Ca, the transient exhibited a similar initial disordering phase (1/tau(1) = 38.5 s(-1)), followed by a recovery phase (1/tau(2) = 8.33 s(-1)) of substantial amplitude, corresponding to the forward rotation and ordering of the lever arm. A standard kinetic model was used to fit these data, revealing rate constants consistent with those previously determined by other methods. Surprisingly, a comparison of the EPR transients with force transients reveals that the rate of force development (91 s(-1)) is faster than the rate of the forward lever arm rotation (8 s(-1)). This observed relationship between the kinetics of the lever arm rotation and transient force development in muscle provides new insight into how myosin both generates and responds to muscle force.
我们利用自旋标记扇贝肌的电子顺磁共振（EPR），结合笼形ATP的激光闪光光解，来解析瞬态力产生过程中肌球蛋白轻链结构域（LCD）的毫秒级旋转转变。我们之前利用EPR解析了LCD的两种不同取向[贝克，J.E.，布吕斯特 - 马舍尔，I.，拉马钱德兰，S.，拉孔特，L.E.，以及托马斯，D.D.（1998年）《美国国家科学院院刊》95，2944 - 2949]，将这些结构状态与肌动蛋白 - 肌球蛋白ATP酶反应中的生化状态相关联，并表明这两种LCD取向之间稳态分布的微小变化（即净杠杆臂旋转）与肌肉中的力产生相关。在本文所呈现的研究中，我们测量了在笼形ATP闪光光解后，肌肉中这种取向分布的毫秒级变化（即两种LCD取向之间的转变速率），分别在有钙和无钙的情况下进行测量。在无钙情况下获得的瞬态主要由一个快速（1/τ(1) = 37 s⁻¹）的无序转变主导，从僵直状态下的单一取向转变为松弛状态下分离的横桥所观察到的双峰取向分布（即杠杆臂旋转的逆转），随后是一个恢复阶段（1/τ(2) = 2.4 s⁻¹），幅度非常小（参与的头部比例小）。在有钙的情况下，瞬态表现出类似的初始无序阶段（1/τ(1) = 38.5 s⁻¹），随后是一个幅度较大的恢复阶段（1/τ(2) = 8.33 s⁻¹），对应于杠杆臂的向前旋转和有序化。使用一个标准动力学模型来拟合这些数据，得到的速率常数与之前通过其他方法确定的一致。令人惊讶的是，将EPR瞬态与力瞬态进行比较发现，力发展的速率（91 s⁻¹）比向前杠杆臂旋转的速率（8 s⁻¹）更快。在肌肉中观察到的杠杆臂旋转动力学与瞬态力发展之间的这种关系，为肌球蛋白如何产生和响应肌肉力提供了新的见解。
