Fluorescence polarization transients from rhodamine isomers on the myosin regulatory light chain in skeletal muscle fibers
Fluorescence polarization transients from rhodamine isomers on the myosin regulatory light chain in skeletal muscle fibers
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DOI：
10.1016/s0006-3495(98)78016-6
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发表时间：
1998-06-01
影响因子：
3.4
通讯作者：
Goldman, YE
中科院分区：
文献类型：
--
作者：
Hopkins, SC;Sabido-David, C;Goldman, YE
利用荧光偏振观察了骨骼肌纤维中与肌球蛋白头结合的两种罗丹明探针的取向变化。用碘乙酰氨基四甲基罗丹明（IATR）的5-或6-异构体在Cys（108）标记鸡胗肌球蛋白调节轻链（RLC）。标记的RLC（称为Cys（108-5）或Cys(108-6)）与兔腰肌单皮纤维中的内源性RLC交换。采用三个独立的荧光偏振比来确定探针偶极子相对于光纤轴的静态角分布和探针在荧光寿命纳秒时间尺度上的运动程度。我们使用纤维长度的阶跃变化来部分同步肌球蛋白交叉桥的生化、结构和机械状态之间的转变。主动收缩时的释放使Cys（108-6）偶极子偏离纤维轴。当释放量超过3nm /半肌节（h / s）时，这种反应达到饱和。主动收缩中的拉伸导致偶极子向纤维轴倾斜，拉伸达到7nm /h时没有饱和的迹象。这些对长度变化的非线性响应与类似于90%的探针在施加长度变化时不倾斜和10%的探针倾斜的划分是一致的。响应分数以7.5 nm/h / s的速度倾斜约30度。释放并穿过垂直于光纤轴的平面以获得更大的释放。严格倾斜的Cys（108-6）偶极子远离纤维轴伸展，这与主动收缩的响应相反。当纤维在Ca2+存在下被囚禁的ATP光解激活时，从严格型到主动型对拉伸的反应的转变先于主要力量的发展。Cys（108-6）在低离子强度（20 mM）弛豫溶液中的极化比与弛豫强度（200 mM）和严格强度的组合相容，但对长度变化的响应为活性型。Cys（108-6）偶极子的纳秒运动被限制在一个类似于20度半角的锥体上，Cys（108-5）偶极子的纳秒运动被限制在一个类似于25度半角的锥体上。这些值在松弛、主动收缩和严格之间变化不大。Cys（108-5）在主动收缩时，拉伸和释放时纤维轴的倾斜幅度都很小，但在收缩时，纤维轴的倾斜幅度要大得多。两种探针异构体之间以及主动收缩和严格性之间对长度步长的响应的显着差异表明，RLC在这两种状态之间经历了很大的角度变化（类似于60度）。这种运动很可能是RLC相对于纤维轴的倾斜和RLC围绕其自身轴的扭曲的结合。
Fluorescence polarization was used to examine orientation changes of two rhodamine probes bound to myosin heads in skeletal muscle fibers. Chicken gizzard myosin regulatory light chain (RLC) was labeled at Cys(108) with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLC (termed Cys(108-5) or Cys(108-6)) was exchanged for the endogenous RLC in single, skinned fibers from rabbit psoas muscle. Three independent fluorescence polarization ratios were used to determine the static angular distribution of the probe dipoles with respect to the fiber axis and the extent of probe motions on the nanosecond time scale of the fluorescence lifetime. We used step changes in fiber length to partially synchronize the transitions between biochemical, structural, and mechanical states of the myosin cross-bridges. Releases during active contraction tilted the Cys(108-6) dipoles away from the fiber axis. This response saturated for releases beyond 3 nm/half-sarcomere (h.s.). Stretches in active contraction caused the dipoles to tilt toward the fiber axis, with no evidence of saturation for stretches up to 7 nm/h.s. These nonlinearities of the response to length changes are consistent with a partition of similar to 90% of the probes that did not tilt when length changes were applied and 10% of the probes that tilted. The responding fraction tilted similar to 30 degrees for a 7.5 nm/h.s. release and traversed the plane perpendicular to the fiber axis for larger releases. Stretches in rigor tilted Cys(108-6) dipoles away from the fiber axis, which was the opposite of the response in active contraction. The transition from the rigor-type to the active-type response to stretch preceded the main force development when fibers were activated from rigor by photolysis of caged ATP in the presence of Ca2+. Polarization ratios for Cys(108-6) in low ionic strength (20 mM) relaxing solution were compatible with a combination of the relaxed (200 mM ionic strength) and rigor intensities, but the response to length changes was of the active type. The nanosecond motions of the Cys(108-6) dipole were restricted to a cone of similar to 20 degrees half-angle, and those of Cys(108-5) dipole to a cone of similar to 25 degrees half-angle. These values changed little between relaxation, active contraction, and rigor. Cys(108-5) showed very small-amplitude tilting toward the fiber axis for both stretches and releases in active contraction, but much larger amplitude tilting in rigor. The marked differences in these responses to length steps between the two probe isomers and between active contraction and rigor suggest that the RLC undergoes a large angle change (similar to 60 degrees) between these two states. This motion is likely to be a combination of tilting of the RLC relative to the fiber axis and twisting of the RLC about its own axis.