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

利用强同步x射线衍射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，并对其数据进行了分析。所得结果如下：(1)基于ccd二维探测器的时间分辨x射线衍射显示，肌钙蛋白相关的第一次子午反射（TN1）在刺激开始时强度增加到约140%，然后随着力的发展而降低，达到静息值以下的水平。TN2强度随张力的发展呈平行下降趋势，TN3强度随张力的发展呈上升趋势。在非重叠长度的肌肉激活时，TN1的强度在刺激开始时增加了约40%，但在平台期保持在约140%的水平。径向反射宽度随力的增大而增大。校正径向宽度后，TN1强度衰减，在初始增加后基本停留在静止水平。对TN1-TN4强度变化的建模分析表明，在激活时，伸长结构域相对于TN分子的球形结构域移动，增加了两个结构域之间的重叠，并且在平台力发展水平期间，两个结构域之间的重叠向静息状态减少。结果表明，与肌肉收缩调控机制相关的TN分子结构变化发生在激活和发力两个过程中。(2)利用低发射同步电子束，研究了拉伸作用下收缩肌肉的二维x射线衍射图中的第一个肌动蛋白层线，阐明了肌动蛋白细丝的可伸性和扭转变化之间的密切关系。拉伸收缩肌时，第一层线的相对间距变化约为0.90%，过度拉伸肌肉激活时，第一层线的相对间距变化为-1.38%，证实了伸展性与左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从静息状态下的80单位/37转对称，到激活状态下的106单位/49转对称，再到发力状态下的54单位/25转对称。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Ca2+开关，在收缩过程中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高空间分辨率的二维x射线衍射图，并测量了肌动蛋白层线的强度。37 nm反射对应于三阶肌动蛋白经向反射。利用7.1 nm以上层线的高角度强度数据，从Holmes的原子丝模型（1990）开始，对包含肌钙蛋白分子核心结构域的肌动蛋白丝进行建模分析。在建模中，在不破坏二级结构的情况下，将丝中的肌动蛋白单体细分为16段，并作为刚体移动。寻找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。通过对Holmes模型的一些修改，确定了最适合肌动蛋白丝的静息模型。肌钙蛋白核心结构域结合在丝中肌动蛋白的亚结构域1和2上。结果表明，肌动蛋白单体的每四个亚结构域以及肌钙蛋白核心结构域部分在收缩过程中发生了小的运动。移动最大的是子域2；它向丝轴移动了约0.5 nm。TNC部分向肌动蛋白丝外侧移动。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