MODELLING THE INTERFACE BETWEEN ACTIN AND MYOSIN ON ACTIVE MUSCLE

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 先前的工作人员基于分离蛋白质的组合晶体学和电子显微镜数据，提出了肌球蛋白头与肌动蛋白对接的高分辨率模型（Mendelson 和 Morris，1997 PNAS 94：8533；Holmes 等人 2003 Nature 425：423）。另一组还声称，力产生的第一步与肌球蛋白-肌动蛋白界面的重新排列有关，随后是杠杆臂倾斜，并且它与温度相关（Ferenczi et al. 2005 Structure 13:131）。这项工作的目标是收集来自肌肉的小角 X 射线散射 (SAXS) 数据，这些数据可用于检查体内肌动蛋白对接模型的预测以及肌球蛋白 - 肌动蛋白界面是否存在温度依赖性重排。 为此，图案中最敏感的反射是肌动蛋白单体沿肌动蛋白丝规则重复而产生的 2.73 nm 经向反射，该反射在肌球蛋白附着到肌动蛋白上时改变其强度。初步建模表明，反射强度受杠杆臂倾斜的影响很小，但对肌动蛋白和肌球蛋白催化域的相对轴向位置高度敏感。 2D 图案将从静止时和不同温度（4 至 17℃）等长收缩期间的肌肉中采集，倒易空间高达 0.5 nm-1，以收集基于肌动蛋白的 2.73 nm 经向反射以及 5.9 nm 和 5.1 nm 层线，也受到肌球蛋白附着到肌动蛋白的影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Previous workers have proposed high resolution models for the docking of the myosin heads on actin on the basis of combined crystallographic and electron microscopy data from isolated proteins (Mendelson and Morris, 1997 PNAS 94:8533; Holmes et al. 2003 Nature 425:423). Another group also claims that the first step in the force generation is associated with a rearrangement of the myosin-actin interface, followed by the lever arm tilt, and that it is temperature-dependent (Ferenczi et al. 2005 Structure 13:131). The goal of this work is to collect small angle X-ray scattering (SAXS) data from muscle that may be used to check in vivo the prediction of the models for the acto-myosin docking and whether there is a temperature-dependent rearrangement of the myosin-actin interface. For this purpose, the most sensitive reflection in the pattern is the 2.73nm meridional reflection arising from the regular repeat of the actin monomers along the actin filament, which changes its intensity upon myosin attachment to actin. Preliminary modelling has shown that the reflection intensity is little influenced by the lever arm tilt but it is highly sensitive to the relative axial position of actin and catalytic domain of myosin. 2D patterns will be taken from muscle at rest and during isometric contraction at different temperatures (4 to 17¿C) up to 0.5 nm-1 in reciprocal space, in order to collect the actin-based 2.73nm meridional reflection and the 5.9nm and 5.1nm layer lines, also influenced by myosin attachment to actin.