Ajtai K, Toft D J, Burghardt T P
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905.
Biochemistry. 1994 May 10;33(18):5382-91. doi: 10.1021/bi00184a005.
The angular distribution of myosin cross-bridges in muscle fibers was investigated in four physiological states using a multiple probe analysis of varied extrinsic probes of the cross-bridge [Burghardt & Ajtai (1994) Biochemistry (preceding paper in this issue)]. The analysis combines data of complementary techniques from different probes giving the highest possible angular resolution. Four extrinsic probes of the fast reactive sulfhydryl (SH1) on myosin subfragment 1 (S1) were employed. Electron paramagnetic resonance (EPR) spectra from paramagnetic probes, deuterium- and 15N-substituted for greater sensitivity to orientation, on S1 were measured when the protein was freely tumbling in solution and when it was decorating muscle fibers. The EPR spectra from labeled S1 tumbling in solution were measured at X- and Q-band microwave frequencies to uniquely specify the orientation of the probe relative to the S1 principal hydrodynamic frame. The EPR spectra from labeled S1 decorating muscle fibers in rigor and in the presence of MgADP were measured at X-band and used in the multiple probe analysis of cross-bridge orientation. The time-resolved fluorescence anisotropy decay (TRFAD) of fluorescent probes on S1 was measured when the protein was freely tumbling in solution, and fluorescence polarization (FP) intensities from fluorescent probes modifying SH1 in intact muscle fibers were measured for fibers in rigor, in the presence of MgADP, in isometric contraction, and in relaxation at low ionic strength. The TRFAD measurements limit the range of possible orientations of the probe relative to the S1 principal hydrodynamic frame. The FP intensity measurements were used in the multiple probe analysis of cross-bridge orientation. The combination of the EPR and FP data determined a highly resolved cross-bridge angular distribution in rigor, in the presence of MgADP, in isometric contraction, and in relaxation at low ionic strength. These findings confirm earlier observations of a rigid body rotation of the SH1 region in the myosin head group upon physiological state changes and indicate the path and extent of cross-bridge rotation during contraction. The rotation of the cross-bridge is visualized with computer-generated space-filling models of actomysin in six states of the contraction cycle.
利用对肌球蛋白横桥的多种外在探针进行多重探针分析，研究了肌肉纤维中肌球蛋白横桥在四种生理状态下的角分布[Burghardt & Ajtai (1994) Biochemistry（本期之前的论文）]。该分析结合了来自不同探针的互补技术数据，给出了尽可能高的角分辨率。使用了四种针对肌球蛋白亚片段1（S1）上快速反应性巯基（SH1）的外在探针。当蛋白质在溶液中自由翻滚以及在修饰肌肉纤维时，测量了来自顺磁探针、氘代和15N取代以提高对取向敏感性的S1上的电子顺磁共振（EPR）光谱，这些顺磁探针位于S1上。在X波段和Q波段微波频率下测量了溶液中翻滚的标记S1的EPR光谱，以唯一确定探针相对于S1主要流体动力学框架的取向。在X波段测量了处于僵直状态以及存在MgADP时修饰肌肉纤维的标记S1的EPR光谱，并将其用于横桥取向的多重探针分析。当蛋白质在溶液中自由翻滚时，测量了S1上荧光探针的时间分辨荧光各向异性衰减（TRFAD），并测量了在僵直状态、存在MgADP、等长收缩以及低离子强度松弛状态下完整肌肉纤维中修饰SH1的荧光探针的荧光偏振（FP）强度。TRFAD测量限制了探针相对于S1主要流体动力学框架的可能取向范围。FP强度测量用于横桥取向的多重探针分析。EPR和FP数据的结合确定了在僵直状态、存在MgADP、等长收缩以及低离子强度松弛状态下高度解析的横桥角分布。这些发现证实了早期关于肌球蛋白头部基团中SH1区域在生理状态变化时发生刚体旋转的观察结果，并表明了收缩过程中横桥旋转的路径和程度。利用计算机生成的肌动球蛋白在收缩周期六个状态下的空间填充模型，直观显示了横桥的旋转。
