Development of an Outward Proton Pumping Rhodopsin with a New Record in Thermostability by Means of Amino Acid Mutations
Development of an Outward Proton Pumping Rhodopsin with a New Record in Thermostability by Means of Amino Acid Mutations
复制标题
通过氨基酸突变开发出具有热稳定性新记录的外向质子泵视紫红质
DOI：
10.1021/acs.jpcb.1c08684
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Murata Takeshi
中科院分区：
文献类型：
--
作者：
Yasuda Satoshi;Akiyama Tomoki;Kojima Keiichi;Ueta Tetsuya;Hayashi Tomohiko;Ogasawara Satoshi;Nagatoishi Satoru;Tsumoto Kouhei;Kunishima Naoki;Sudo Yuki;Kinoshita Masahiro;Murata Takeshi
我们已经开发了一种方法来确定进一步的热稳定性突变的内在热稳定性膜蛋白。该方法包括以下步骤：（1）使用我们基于物理的方法鉴定跨膜区中残基的热稳定性单突变（TSSM）;（2）使用经验力场FoldX鉴定水环境中细胞外和细胞内区中残基的TSSM;和（3）组合步骤（1）和（2）中鉴定的TSSM以构建多个突变。以热变性的表观中点温度Tmis = 91.8 °C的嗜热视紫红质为例说明了该方法。先前在步骤（1）中鉴定的TSSM为F90 K、F90 R和Y 91 I，ΔTm分别为5.6 ° C、5.5 ° C和2.9 °C，步骤（2）中鉴定的TSSM为V79 K、T114 D、A115 P和A116 E，ΔTm分别为2.7 ° C、4.2 ° C、2.6 ° C和2.3 °C（Δ Tm表示Tm增加）。本研究构建了F90 K + Y 91 I + T114 D和F90 K + Y 91 I + V79 K + T114 D的三重和四重突变体。这两个突变体的Δ Tm值分别为Δ 11.4和Δ 13.5 °C，其中四突变体的Tm值为Δ 105.3 °C，创造了一类质子泵视紫红质的新记录。它高于本研究之前类中最耐热的嗜木红杆菌视紫红质的Tmov（100.8 °C）。
We have developed a methodology for identifying further thermostabilizing mutations for an intrinsically thermostable membrane protein. The methodology comprises the following steps: (1) identifying thermostabilizing single mutations (TSSMs) for residues in the transmembrane region using our physics-based method; (2) identifying TSSMs for residues in the extracellular and intracellular regions, which are in aqueous environment, using an empirical force field FoldX; and (3) combining the TSSMs identified in steps (1) and (2) to construct multiple mutations. The methodology is illustrated for thermophilic rhodopsin whose apparent midpoint temperature of thermal denaturationTmis ∼91.8 °C. The TSSMs previously identified in step (1) were F90K, F90R, and Y91I with ΔTm∼5.6, ∼5.5, and ∼2.9 °C, respectively, and those in step (2) were V79K, T114D, A115P, and A116E with ΔTm∼2.7, ∼4.2, ∼2.6, and ∼2.3 °C, respectively (ΔTmdenotes the increase inTm). In this study, we construct triple and quadruple mutants, F90K+Y91I+T114D and F90K+Y91I+V79K+T114D. The values of ΔTmfor these multiple mutants are ∼11.4 and ∼13.5 °C, respectively.Tmof the quadruple mutant (∼105.3 °C) establishes a new record in a class of outward proton pumping rhodopsins. It is higher thanTmofRubrobacter xylanophilusrhodopsin (∼100.8 °C) that was the most thermostable in the class before this study.