Further thermo-stabilization of thermophilic rhodopsin from Thermus thermophilus JL-18 through engineering in extramembrane regions

Tomoki Akiyama, Naoki Kunishima, Sayaka Nemoto, Kazuki Kazama, Masako Hirose, Yuki Sudo, Yoshinori Matsuura, Hisashi Naitow, Takeshi Murata

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)301-310
Number of pages10
JournalProteins: Structure, Function and Bioinformatics
Issue number3
Publication statusPublished - Mar 2021


  • differential scanning calorimetry
  • membrane protein
  • molecular dynamics
  • optogenetics
  • protein stability
  • site-directed mutagenesis

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology


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