Analysis of structure-function relationships on smooth muscle myosin using NMR and X-ray crystallography.

肌球蛋白是一种酶，它与肌动蛋白一起将ATP水解产生的化学能转化为定向机械运动。本研究利用核磁共振技术和X射线晶体学技术，从原子水平上探讨了肌球蛋白的能量转换机制，从<31>平滑肌亚片段1-ADP复合物~ 1 P-NMR谱信号随pH和温度的变化，平滑肌肌球蛋白亚片段1与ADP的相互作用强于骨骼肌肌球蛋白亚片段1与ADP的相互作用。在驱动蛋白和ncd运动域的情况下，发现存在两种不同形式的运动域-ADP复合物。此外，我们还发现驱动蛋白和ncd马达结构域结合ADP的α-磷酸的化学位移存在差异，表明这两种马达结构域结合ADP的α-磷酸的静电或磁微环境不同。我们建立了杆状病毒表达系统，获得了重组鸡肌球蛋白马达结构域及其结晶突变体。我们还发现颈部区域对驱动蛋白ATP酶活性的影响。

Myosin is an enzyme transducing the chemical energy from the hydrolysis of ATP into directed mechanical movement, in conjunction with actin. In this research, we use the NMR, which is a good probe to observe the microenvironments, and X-ray crystallography, which allows us a precise three-dimensional structure determination, to investigate the mechanism of energy conversion by myosin with atomic level.From the pH- and temperature-dependent changes of the signals in the ^<31>P-NMR spectra of smooth muscle sub fragment 1-ADP complex, it is appeared that the interaction between the smooth muscle myosin subfragment 1 and ADP is stronger than that of skeletal muscle myosin subfragment 1 and ADP. In the case of kinesin and ncd motor domains, it was found to exist two different forms of motor domain-ADP complex. In addition, we found a difference between kinesin and ncd motor domains in the chemical shift of the a-phosphate of bound ADP, indicating that the electrostatic or magnetic microenvironments of this site of these motor domains differed from each other.We established the baculovirus expression system to obtain the recombinant chicken gizzard smooth muscle myosin motor domain as well as mutants for crystallization. We also found that the role of the neck region on the ATPase activity of kinesin.