核磁共振法研究大肠杆菌细胞内La^(3+)与钙调蛋白的相互作用

Study on Interaction of La^(3+) with Calmodulin by Cellular NMR Methods

应用in-cell和cell free核磁共振(NMR)方法分别研究了柠檬酸镧与大肠杆菌细胞内15N标记的钙调蛋白(CaM)以及La3+和大肠杆菌无细胞提取液(Cell free extract,CFE)的相互作用。将所得1H-15N HSQC NMR谱与纯化过的以不同组成比结合Ca/La的CaM的1H-15N HSQC NMR谱比较,发现在细胞内因胞内环境拥挤,蛋白运动能力下降,使CaM的NMR信号较少且峰宽增加。而柠檬酸镧可以透过细胞膜进入细胞,并与胞内的CaM结合。无细胞提取液的NMR谱显示,加镧之前细胞内CaM主要以Ca结合态存在,而加入La3+可能形成Ca2La2CaM混合金属配合物。这和无细胞的化学体系的实验结果一致。实验结果显示,细胞水平NMR方法可用于研究稀土以至其他金属配合物与细胞的相互作用如何与细胞内钙调蛋白作用,以及有关的生物功能。

The interactions of La^3＋ and calmodulin （CAM） in the living cells were investigated using incell NMR method by comparing the ^1H-^15N HSQC NMR spectra of CaM in E. coli cells and cell free extract（CFE）with the purified CaM species. It was observed that the NMR spectra of in-cell samples exhibited fewer signals and broader line width due to the low mobility of proteins in crowed cellular surroundings. The change of the NMR spectra suggested that La^3＋-citrate complexes could permeate into E. coli ceils and bind to CaM. Studies with the CFE samples suggested that CaM exists as Ca-binding form in the living ceils while La^3＋ may form the hybrid complex of Ca/La/CaM as we proposed previously. The results suggested that it is of great potential to study the biological interaction of lanthanide ions with CaM by use of the cellular NMR methods.