A methodology for creating thermostabilized mutants of G‐protein coupled receptors by combining statistical thermodynamics and evolutionary molecular engineering
A methodology for creating thermostabilized mutants of G‐protein coupled receptors by combining statistical thermodynamics and evolutionary molecular engineering
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结合统计热力学和进化分子工程创建 G 蛋白偶联受体热稳定突变体的方法
DOI：
10.1002/pro.4404
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发表时间：
2022
期刊：
影响因子：
8
通讯作者：
Murata Takeshi
中科院分区：
文献类型：
--
作者：
Sugaya Kanna;Yasuda Satoshi;Sato Shingo;Sisi Chen;Yamamoto Taisei;Umeno Daisuke;Matsuura Tomoaki;Hayashi Tomohiko;Ogasawara Satoshi;Kinoshita Masahiro;Murata Takeshi
我们构建了一种用于热稳定处于非活性状态的G蛋白偶联受体（GPCR）的方法，该受体的野生型（WT）结构仅通过多个氨基酸突变而未知，而没有配体结合。它是我们最近开发的基于统计热力学和定点饱和诱变的理论的结合，定点饱和诱变是进化分子工程中经常采用的方法。首先，使用同源建模预测WT结构。其次，通过我们的统计热力学理论使用适当建模的突变体结构来确定关键残基。预计关键残基的19种不同单突变中的许多突变会产生显著更高的稳定性。第三，我们不仅对关键残基进行突变，而且对侧链与关键残基的侧链接近的几个残基进行突变。然后通过引入定点饱和突变有效地探索整个突变空间，并使用小智能和全自动单管重组方法构建基因（突变体）文库。每个突变体在大肠杆菌细胞中表达，并使用荧光筛选技术分选出高度稳定的突变体。该方法用于5-羟色胺2A受体（5-HT 2AR），以稳定其非活性状态。我们可以鉴定出一种双突变体，其热变性的表观中点温度比之前报道的热稳定双突变体高约8.9°C，比WT高15°C以上。此外，它对螺哌隆（一种先前被证明可稳定非活性状态下5-HT 2AR的拮抗剂）表现出更高的结合亲和力。
We constructed a methodology for thermostabilizing a G‐protein coupled receptor (GPCR) in the inactive state whose wild‐type (WT) structure is unknown solely by multiple amino‐acid mutations without the ligand binding. It is a combination of our recently developed theory based on statistical thermodynamics and site‐directed saturation mutagenesis, a method often employed in evolutionary molecular engineering. First, the WT structure is predicted using the homology modeling. Second, a key residue is determined by our statistical‐thermodynamics theory using suitably modeled mutant structures. Many of 19 different single mutations for the key residue are expected to produce significantly higher stabilization. Third, we undertake to mutate not only the key residue but also a few more residues whose side chains are close to the side chain of the key residue. The whole mutational space is then efficiently explored by introducing site‐directed saturation mutations, and a gene (mutant) library is constructed using the small‐intelligent and fully automatic single‐tube recombination methods. Each mutant is expressed inEscherichia colicells, and highly stabilized mutants are sorted out using a fluorescence‐screening technique. The methodology was illustrated for the serotonin 2A receptor, 5‐HT2AR, for stabilizing its inactive state. We could identify a double mutant whose apparent midpoint temperature of thermal denaturation is higher than that of a thermostabilized double mutant previously reported by ~8.9°C and that of the WT by over 15°C. Moreover, it exhibits higher binding affinity for spiperone, an antagonist which was previously proved to stabilize 5‐HT2AR in the inactive state.
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影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
嗜热视紫红质和黄视紫红质热稳定性差异较大的统计热力学
DOI：
--
发表时间：
2018
期刊：
影响因子：
--
作者：
Satoshi Yasuda;Tomohiko Hayashi;Yuta Kajiwara;Takeshi Murata;and Masahiro Kinoshita
通讯作者：
and Masahiro Kinoshita
影响因子：
5.6
作者：
Kinoshita M
通讯作者：
Kinoshita M
影响因子：
56.9
作者：
V. Cherezov;D. Rosenbaum;M. Hanson;S. Rasmussen;Foon Sun Thian;T. S. Kobilka;Hee-Jung Choi;P. Kuhn-P
通讯作者：
V. Cherezov;D. Rosenbaum;M. Hanson;S. Rasmussen;Foon Sun Thian;T. S. Kobilka;Hee-Jung Choi;P. Kuhn-P
影响因子：
5.7
作者：
Chun, Eugene;Thompson, Aaron A.;Liu, Wei;Roth, Christopher B.;Griffith, Mark T.;Katritch, Vsevolod;Kunken, Joshua;Xu, Fei;Cherezov, Vadim;Hanson, Michael A.;Stevens, Raymond C.
通讯作者：
Stevens, Raymond C.