期刊
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
卷 89, 期 3, 页码 301-310出版社
WILEY
DOI: 10.1002/prot.26015
关键词
differential scanning calorimetry; membrane protein; molecular dynamics; optogenetics; protein stability; site‐ directed mutagenesis
资金
- Japan Society for the Promotion of Science (JSPS) [18H05425, 19H05396]
- Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)
- Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)
- Japan Agency for Medical Research and Development (AMED)
- Grants-in-Aid for Scientific Research [18H05425, 19H05396] Funding Source: KAKEN
In this study, thermo-stabilization of the thermophilic rhodopsin from Thermus thermophilus JL-18 as a model membrane protein was achieved by introducing ten single mutations in the extramembrane regions. Experimental characterization and molecular dynamics simulations confirmed that four mutations improved thermal stability, highlighting the potential of this approach for improving the production of membrane proteins for structural studies.
It is known that a hyperthermostable protein tolerable at temperatures over 100 degrees 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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据