Selection and analyses of variants of a designed protein suggest importance of hydrophobicity of partially buried sidechains for protein stability at high temperatures
Selection and analyses of variants of a designed protein suggest importance of hydrophobicity of partially buried sidechains for protein stability at high temperatures
复制标题
对设计蛋白质变体的选择和分析表明部分掩埋侧链的疏水性对于蛋白质高温稳定性的重要性
DOI：
10.1002/pro.3643
复制
发表时间：
2019-08-01
期刊：
影响因子：
8
通讯作者：
Liu, Haiyan
中科院分区：
文献类型：
--
作者：
Han, Mingjie;Liao, Sanhui;Liu, Haiyan
计算设计的高稳定性蛋白质提供了标本，除了天然蛋白质的序列结构稳定性关系的研究在非常高端的蛋白质稳定性谱。E_1r26是我们先前使用基于骨架的氨基酸使用调查（ABACUS）序列设计程序设计的蛋白质，其解链温度高于110摄氏度，比天然硫氧还蛋白的解链温度高50摄氏度以上，天然硫氧还蛋白的骨架（PDB ID）已被用作设计目标。使用实验选择的方法，我们得到的变体E_1r26保持折叠，但稳定性降低，包括一个其展开温度和变性胍浓度是类似的1r26。突变体以一定程度的协同性展开。它的结构解决了X射线晶体学同意1r26的均方根偏差为1.3埃，增加支持ABACUS方法的准确性。中间突变体的分析表明，由极性残基（苏氨酸和丝氨酸，分别）的两个部分掩埋的疏水残基（异亮氨酸和亮氨酸）的取代是负责的展开温度的急剧变化。这表明，位于刚性二级结构区域的突变的影响，而不是那些在环，可以很好地预测通过ABACUS突变能量分析。结果还表明，涉及中间掩埋侧链的疏水效应对于蛋白质在高温下的稳定性至关重要。
Computationally designed proteins of high stability provide specimen in addition to natural proteins for the study of sequence-structure stability relationships at the very high end of protein stability spectrum. The melting temperature of E_1r26, a protein we previously designed using the A Backbone-based Amino aCid Usage Survey (ABACUS) sequence design program, is above 110 degrees C, more than 50 degrees C higher than that of the natural thioredoxin protein whose backbone (PDB ID ) has been used as the design target. Using an experimental selection approach, we obtained variants of E_1r26 that remain folded but are of reduced stability, including one whose unfolding temperature and denaturing guanidine concentration are similar to those of 1r26. The mutant unfolds with a certain degree of cooperativity. Its structure solved by X-ray crystallography agrees with that of 1r26 by a root mean square deviation of 1.3 angstrom, adding supports to the accuracy of the ABACUS method. Analyses of intermediate mutants indicate that the substitution of two partially buried hydrophobic residues (isoleucine and leucine) by polar residues (threonine and serine, respectively) are responsible for the dramatic change in the unfolding temperature. It is suggested that the effects of mutations located in rigid secondary structure regions, but not those in loops, may be well predicted through ABACUS mutation energy analysis. The results also suggest that hydrophobic effects involving intermediately buried sidechains can be critically important for protein stability at high temperatures.