东亚钳蝎神经毒素BmK CT三维结构分子建模及晶体生长条件的初步筛选

东亚钳蝎是一类广泛分布于我国且具有重要药用价值的野生资源.其氯离子通道神经毒素(BmK CT)对人脑神经胶质瘤细胞具有靶向特异性的抑制作用.利用SWISS-MODEL服务器对BmK CT进行蛋白质原子结构的同源建模,利用Insight II软件包对建模结果做能量最小化和分子动力学优化,获得最佳候选模型.三维结构对比分析表明,BmK CT与以色列蝎氯通道毒素(Cltx)三维结构极为相似,均由一个α-helix和两个反向平行的β-sheet构成,但BmK CT属于βαβ型,而Cltx属于αββ型.表面静电势分析表明,BmK CT和Cltx分子表面分布着大量正电势,暗示两分子功能相似,且其中的碱性氨基酸及产生的正电势对其与受体结合起决定作用.采用晶体生长试剂盒(Crystal Screening Kit)中的51个缓冲液配方筛选BmK CT的结晶条件,在7号配方(1.4 mol/L 乙酸钠,0.1 mol/L 甲次砷酸钠,pH 6.51中筛选到柱状六边形晶体.

Prediction of Three Dimensional Structure and Filtration of Crystallization Condition of BmK CT

BmK CT,one of the key toxin from Buthus matensii Karsch,has been shown to bind specifically to glioma cell surface as a specific chloride channel blocker. In this study, three dimensional structure of BmK CT was modeled and the crystallization condition of BmK CT was filtrated. Prediction of three dimensional structure of BmK CT was performed by SWISS-MODEL workspace and the quality of generated mode was further examined by energy minimization and molec-ular dynamics (MD) simulation using Insight II 2000 software package. Crystallization of BmK CT was performed using the hanging-drop method. 51 conditions (Crystal Screening Kit, Hampton Research) were used to get good quality and well repeating crystals. The structure of BmK CT and Cltx consisted of a small three-stranded antiparallel β-sheet packed against an α-helix. But BmK CT belonged to βαβ type and Cltx αββ type. The calculated electrostatic potential surface around the BmK CT and Cltx molecule were found to be mainly positive, which suggested that this clustering of positively charged residues and the strong positive field they produce might well have important implications on the interaction with the receptors. The results showed that hexagonal column crystal could be obtained from a condition containing 1.4 mol/L sodium acetate,0.1 mol/L Na cacodylate,pH 6.51,at 4℃ and the protein was dissolved in ddH_2O.