Barnase早期热变性位点的动力学模拟研究  

Early denaturation sites in thermal unfolding of Barnase as revealed by molecular dynamics simulation

作　　者：傅毅[1,2] 吴小俊[1] 

机构地区：[1]江南大学物联网工程学院,江苏无锡214122 [2]无锡城市学院电子信息工程系,江苏无锡214153

出　　处：《计算机与应用化学》2013年第11期1305-1309,共5页Computers and Applied Chemistry

摘　　要：解淀粉芽孢杆菌核糖核酸酶Barnase是解淀粉芽孢杆菌(Bacillus amyloliquefaciens)产生的一种I2kD的胞外小分子RNA酶(Ribonuclease,RNase),在农业、医疗、制药等领域有广泛的应用前景。该酶包含3个螺旋,5个折叠,由于没有二硫键限制其解折叠状态,非常适合作为模型系统研究蛋白质的折叠问题。使用分子动力学模拟对Bamase的热解折叠进行研究,通过对Bamase分别在5个不同温度下运行动力学模拟,研究在热解折叠路径上蛋白质的结构与稳定性的关系,通过分析不同温度下蛋白质的结构变化来寻找其解折叠重要区域和蛋白质不同区域的热稳定性的差别。实验发现在温度400 K的时候可以观察到蛋白质开始发生解折叠,高温下Barnase的初始解折叠发生在N端;酶中Ioopl和loop2的不稳定使得疏水核2相对快速地变性;α3螺旋比其他2个α螺旋要开始解折叠的早:疏水核3中的β折叠的破裂开始于β1凸起附近和β1和β5的边缘部分。模拟结果显示蛋白质的稳定性并不是均衡的分布于蛋白质之中,在蛋白质全局结构被破坏前,早期变性位点所位于的局部结构就开始发生解折叠,因此通过适当的氨基酸来替换这些位点,则可能设计出更稳定的满足需要的突变体。Bacillus amyloliquefaciens ribonuclease barnase（RNase Ba） is a 12 kD small extracellular ribonuclease. It has broad application prospects in agriculture, clinical medicine, pharmaceutical, etc. Barnase consists of three a-helices and five β-sheets and are stabilized by three hydrophobic cores in the native structure. There are no disulfide linkages to constrain the unfolded state, this protein is a particularly good system for folding study. In this study, we have performed molecular dynamics simulation of Barnase at high temperatures. And then, we discuss the structure-stability relationship with focus on possible differences in the thermal unfolding pathway. At the same time, we have especially analyzed molecular structure at different temperatures, in order to search for structurally important regions, which could explain the difference in thermal stability of the protein during the unfolding process. The protein begins to unfold at 400 K simulation temperature. Distortion of the enzyme is initiated in the N-terminal and loops. Hydrophobic core 2 denatures relatively rapidly, followed by a3-helix and hydrophobic core 3. The β-sheet disruption starts near β-bulge at residues 53 and 54 and at the edges β-strands 1 and 5. The results show clearly that the stability of the protein is not evenly distributed over the whole structure. These results suggest some local structures which contain early denaturation sites are disrupted prior to global structure disruption. Identification of denaturation sites suggests that appropriate amino acid replacements at these sites may lead to the design and development of more stable Barnase mutants.

关 键 词：解淀粉芽孢杆菌核糖核酸酶 解折叠 热稳定 氢键 分子动力学模拟 

分 类 号：TP391.9[自动化与计算机技术—计算机应用技术]