Rational thermostabilisation of four-helix bundle dimeric de novo proteins
Abstract The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. T...
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Nature Portfolio
2021-04-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-021-86952-2 |
| _version_ | 1818401661602758656 |
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| author | Shin Irumagawa Kaito Kobayashi Yutaka Saito Takeshi Miyata Mitsuo Umetsu Tomoshi Kameda Ryoichi Arai |
| author_facet | Shin Irumagawa Kaito Kobayashi Yutaka Saito Takeshi Miyata Mitsuo Umetsu Tomoshi Kameda Ryoichi Arai |
| author_sort | Shin Irumagawa |
| collection | DOAJ |
| description | Abstract The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (T m). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔT m = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its T m. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest T m (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes. |
| first_indexed | 2024-12-14T07:56:01Z |
| format | Article |
| id | doaj.art-c7e9e6f72c2f486ea2f007ade93eb967 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-14T07:56:01Z |
| publishDate | 2021-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-c7e9e6f72c2f486ea2f007ade93eb9672022-12-21T23:10:33ZengNature PortfolioScientific Reports2045-23222021-04-0111111010.1038/s41598-021-86952-2Rational thermostabilisation of four-helix bundle dimeric de novo proteinsShin Irumagawa0Kaito Kobayashi1Yutaka Saito2Takeshi Miyata3Mitsuo Umetsu4Tomoshi Kameda5Ryoichi Arai6Department of Science and Technology, Graduate School of Medicine, Science and Technology, Shinshu UniversityArtificial Intelligence Research Center, National Institute of Advanced Industrial Science and Technology (AIST)Artificial Intelligence Research Center, National Institute of Advanced Industrial Science and Technology (AIST)Department of Biochemistry and Biotechnology, Faculty of Agriculture, Kagoshima UniversityDepartment of Biomolecular Engineering, Graduate School of Engineering, Tohoku UniversityArtificial Intelligence Research Center, National Institute of Advanced Industrial Science and Technology (AIST)Department of Science and Technology, Graduate School of Medicine, Science and Technology, Shinshu UniversityAbstract The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (T m). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔT m = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its T m. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest T m (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.https://doi.org/10.1038/s41598-021-86952-2 |
| spellingShingle | Shin Irumagawa Kaito Kobayashi Yutaka Saito Takeshi Miyata Mitsuo Umetsu Tomoshi Kameda Ryoichi Arai Rational thermostabilisation of four-helix bundle dimeric de novo proteins Scientific Reports |
| title | Rational thermostabilisation of four-helix bundle dimeric de novo proteins |
| title_full | Rational thermostabilisation of four-helix bundle dimeric de novo proteins |
| title_fullStr | Rational thermostabilisation of four-helix bundle dimeric de novo proteins |
| title_full_unstemmed | Rational thermostabilisation of four-helix bundle dimeric de novo proteins |
| title_short | Rational thermostabilisation of four-helix bundle dimeric de novo proteins |
| title_sort | rational thermostabilisation of four helix bundle dimeric de novo proteins |
| url | https://doi.org/10.1038/s41598-021-86952-2 |
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