X-ray diffraction analysis of structural charges of regulatory proteins and tins in muscle contraction.

利用同步辐射X射线衍射技术测量了肌肉收缩过程中调节蛋白（肌钙蛋白和原肌球蛋白）和肌动蛋白细丝的结构变化，并对数据进行了分析。所得结果如下。（1）在肌肉等长肌力发展过程中，在粗细肌丝完全重叠长度处，采用基于CCD的2D探测器的时间分辨X射线衍射显示，第一肌钙蛋白相关的肌钙蛋白反射（TN 1）在刺激开始时增加其强度至约2.5 μ g/ml。140%，然后随着力量的发展而下降，达到静息值以下的水平。TN 2强度随张力的发展而降低，TN 3强度随张力的发展而升高，并走在张力的前面。在非重叠长度的肌肉激活时，TN 1的强度增加了约10%。40%在刺激开始时，但停留在ca。平台期140%水平 关于我们 力的阶段。但其径向反射宽度随力的发展而增大。校正径向宽度后，TN 1强度衰减，并在初始增加后几乎停留在静息水平。对TN 1 - 100强度变化的模拟分析TN 4表明，在激活时，TN分子的伸长结构域相对于球状结构域移动，增加了两个结构域之间的重叠，在力发展的平台水平期间，两个结构域之间的重叠向静息状态减少。肌肉收缩发生在两个过程中，即激活和力量产生期间。（2）利用低发射率同步辐射光束，通过对拉伸收缩肌肉的二维X射线衍射图中第一肌动蛋白层线的研究，阐明了肌动蛋白细丝的伸展性和扭曲性变化之间的密切关系。第一层线的相对间距变化约为0.05。在施加于收缩肌肉的拉伸期间为0.90%，并且在过度拉伸肌肉的激活时为-1.38%，证实了可伸展性与左旋和右旋遗传螺旋的扭曲性质密切相关。肌动蛋白丝的螺旋对称性从静止状态下的80单位/37圈对称性通过激活时的106单位/49圈改变为力产生状态下的54单位/25圈对称性。这些结果清楚地表明，细肌动蛋白丝本身包括开-关状态，作为Ca ~（2+）开关操作，并且在收缩期间，肌动蛋白丝的伸展性与肌肉中弹性元件的伸展直接相关。（3）在收缩过程中拍摄了具有高空间分辨率的二维X射线衍射图，并测量了肌动蛋白基底层线的强度，直到37 nm的反射，对应于三级肌动蛋白双折射反射。使用7.1nm以上的层线的高角度强度数据，从Holmes的原子丝模型（1990）开始，对包括肌钙蛋白分子的核心结构域的肌动蛋白丝进行建模分析。在不破坏二级结构的前提下，将微丝中的肌动蛋白单体细分为16段，并将其作为刚体进行运动。搜索肌动蛋白丝上肌钙蛋白核心结构域的配置和排列，以获得R因子的最小值。肌动蛋白丝的最佳拟合静止模型是通过对Holmes模型的一些修改来确定的。肌钙蛋白核心结构域在肌丝中结合在肌动蛋白的亚结构域1和2上。结果表明，在收缩过程中，肌动蛋白单体中的每四个亚结构域以及肌钙蛋白核心结构域部分的分布都发生了微小的运动。最大的运动是子域2，它向丝轴移动了约。0.5 nm. TNC部分向肌动蛋白丝的外侧移动。少

Structural changes of the regulatory proteins (troponin and tropomyosin) and the thin actin filaments occurring in the muscle contraction process have been measured by using an intense synchrotron X-ray diffraction, and their data were analyzed. The obtained results are as follows.(1)Time-resolved X-ray diffraction with a CCD-based 2D detector in the process of isometric force development of muscle at the full overlap length of thin and thick filaments showed that the first troponin-associated meridional reflection (TN1) increased its intensity at the onset of stimulation to ca. 140% and then decreased it together with the development of force, reaching the level below the resting value. The TN2 intensity decreased in parallel with the development of tension but the TN3 intensity increased and run ahead the tension. On activation of muscle at the non-overlap length, the intensity of TN1 increased by ca. 40% on the onset of stimulation but stayed at the ca. 140% level during the plateau … More phase of force. However, their radial reflection width increased with the development of force. After the correction of the radial width, the TN1 intensity decayed and stayed almost at the resting level after an initial increase. The modeling analysis of the intensity changes of TN1-TN4 suggested that on activation the elongated domain moved relative to the globular domain of a TN molecule to increase the overlap between the two domains and during the plateau level of force development the overlap between the two domains decreased toward the resting state.The results indicates that the structural changes of the TN molecules which are related to the regulatory mechanism in muscle contraction are occurring in the two processes, namely on activation and during force generation.(2)With the use of a low emittance synchrotron beam, the close relationship between the extensibility and twisting changes of the thin actin filaments was clarified by investigating the first actin-based layer line in the 2D X-ray diffraction patterns from a contracting muscle by the application of stretch. The relative spacing change of the first layer line was ca. 0.90% during stretch applied to contracting muscle and was -1.38% on activation of the overstretched muscle, confirming that the extensibility is closely related with the nature of the twisting of the left-and right-handed genetic helices. The helical symmetry of the actin filament altered from an 80 units/37 turns symmetry in the resting state through a 106 units/49 turns on activation to a 54 units/25 turns symmetry in the force generating state. These results clearly indicate that the thin actin filament itself include the on-off-states, operating as the Ca2+ switch and during contraction the extensibility of the actin filaments is directly related to the extention of the elastic element in muscle.(3)The 2D X-ray diffraction patterns with a high-spatial resolution were taken during contraction, and the intensities of the actin-based layer lines were measured upto thel.37 nm reflection corresponding to the third order actin meridional reflection. Using the higher-angle intensity data of the layer lines above the 7.1 nm, the modeling analysis of the actin filament including the core domain of troponin molecules was performed by starting from the Holmes' atomic filament model (1990). In the modeling the actin monomer in the filament was subdivided into 16 segments without destroying the secondary structures and they were moved as a rigid body. The disposition and arrangement of the troponin core domains on the actin filament were searched to obtain the minimum value of the R-factor. The best-fit resting model of the actin filament was determined with some modification of the Holmes' model. The troponin core domain bound over the subdomains 1 and 2 of actin in the filament. The results indicate that small movements of each four subdomains in the actin monomer as well as the disposition of the troponin core domain portion take place during contraction. The largest movement was the subdomain 2; it moved toward the filament axis by ca. 0.5 nm. The TNC part moved toward the outside of the actin filament. Less