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Further thermo‐stabilization of thermophilic rhodopsin from Thermus thermophilus JL ‐18 through engineering in extramembrane regions
Author(s) -
Akiyama Tomoki,
Kunishima Naoki,
Nemoto Sayaka,
Kazama Kazuki,
Hirose Masako,
Sudo Yuki,
Matsuura Yoshinori,
Naitow Hisashi,
Murata Takeshi
Publication year - 2021
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.26015
Subject(s) - thermus thermophilus , mutant , native state , differential scanning calorimetry , biophysics , molecular dynamics , membrane , membrane protein , mutation , rhodopsin , folding (dsp implementation) , protein tertiary structure , chemistry , crystallography , biology , biochemistry , thermodynamics , physics , gene , computational chemistry , retinal , escherichia coli , electrical engineering , engineering
It is known that a hyperthermostable protein tolerable at temperatures over 100°C can be designed from a soluble globular protein by introducing mutations. To expand the applicability of this technology to membrane proteins, here we report a further thermo‐stabilization of the thermophilic rhodopsin from Thermus thermophilus JL‐18 as a model membrane protein. Ten single mutations in the extramembrane regions were designed based on a computational prediction of folding free‐energy differences upon mutation. Experimental characterizations using the UV‐visible spectroscopy and the differential scanning calorimetry revealed that four of ten mutations were thermo‐stabilizing: V79K, T114D, A115P, and A116E. The mutation‐structure relationship of the TR constructs was analyzed using molecular dynamics simulations at 300 K and at 1800 K that aimed simulating structures in the native and in the random‐coil states, respectively. The native‐state simulation exhibited an ion‐pair formation of the stabilizing V79K mutant as it was designed, and suggested a mutation‐induced structural change of the most stabilizing T114D mutant. On the other hand, the random‐coil‐state simulation revealed a higher structural fluctuation of the destabilizing mutant S8D when compared to the wild type, suggesting that the higher entropy in the random‐coil state deteriorated the thermal stability. The present thermo‐stabilization design in the extramembrane regions based on the free‐energy calculation and the subsequent evaluation by the molecular dynamics may be useful to improve the production of membrane proteins for structural studies.