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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Autores principales:	Shin Irumagawa, Kaito Kobayashi, Yutaka Saito, Takeshi Miyata, Mitsuo Umetsu, Tomoshi Kameda, Ryoichi Arai
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c7e9e6f72c2f486ea2f007ade93eb967
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spelling	oai:doaj.org-article:c7e9e6f72c2f486ea2f007ade93eb9672021-12-02T18:15:25ZRational thermostabilisation of four-helix bundle dimeric de novo proteins10.1038/s41598-021-86952-22045-2322https://doaj.org/article/c7e9e6f72c2f486ea2f007ade93eb9672021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-86952-2https://doaj.org/toc/2045-2322Abstract 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.Shin IrumagawaKaito KobayashiYutaka SaitoTakeshi MiyataMitsuo UmetsuTomoshi KamedaRyoichi AraiNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Shin Irumagawa Kaito Kobayashi Yutaka Saito Takeshi Miyata Mitsuo Umetsu Tomoshi Kameda Ryoichi Arai Rational thermostabilisation of four-helix bundle dimeric de novo proteins
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.
format	article
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
title	Rational thermostabilisation of four-helix bundle dimeric de novo proteins
title_short	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_sort	rational thermostabilisation of four-helix bundle dimeric de novo proteins
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/c7e9e6f72c2f486ea2f007ade93eb967
work_keys_str_mv	AT shinirumagawa rationalthermostabilisationoffourhelixbundledimericdenovoproteins AT kaitokobayashi rationalthermostabilisationoffourhelixbundledimericdenovoproteins AT yutakasaito rationalthermostabilisationoffourhelixbundledimericdenovoproteins AT takeshimiyata rationalthermostabilisationoffourhelixbundledimericdenovoproteins AT mitsuoumetsu rationalthermostabilisationoffourhelixbundledimericdenovoproteins AT tomoshikameda rationalthermostabilisationoffourhelixbundledimericdenovoproteins AT ryoichiarai rationalthermostabilisationoffourhelixbundledimericdenovoproteins
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Rational thermostabilisation of four-helix bundle dimeric de novo proteins

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