Rational thermostabilisation of four-helix bundle dimeric de novo proteins

Shin Irumagawa; Kaito Kobayashi; Yutaka Saito; Takeshi Miyata; Mitsuo Umetsu; Tomoshi Kameda; Ryoichi Arai

doi:10.1038/s41598-021-86952-2

Rational thermostabilisation of four-helix bundle dimeric de novo proteins

Sci Rep. 2021 Apr 6;11(1):7526. doi: 10.1038/s41598-021-86952-2.

Authors

Shin Irumagawa^{1

2

3}, Kaito Kobayashi⁴, Yutaka Saito^{4

5

6}, Takeshi Miyata⁷, Mitsuo Umetsu⁸, Tomoshi Kameda⁴, Ryoichi Arai^{9

10

11}

Affiliations

¹ Department of Science and Technology, Graduate School of Medicine, Science and Technology, Shinshu University, Ueda, Nagano, 386-8567, Japan.
² Department of Biomolecular Innovation, Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Matsumoto, Nagano, 390-8621, Japan.
³ Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, 386-8567, Japan.
⁴ Artificial Intelligence Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, 135-0064, Japan.
⁵ AIST-Waseda University Computational Bio Big-Data Open Innovation Laboratory (CBBD-OIL), Tokyo, 169-8555, Japan.
⁶ Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, 277-8561, Japan.
⁷ Department of Biochemistry and Biotechnology, Faculty of Agriculture, Kagoshima University, Kagoshima, 890-0065, Japan.
⁸ Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, 980-8579, Japan.
⁹ Department of Science and Technology, Graduate School of Medicine, Science and Technology, Shinshu University, Ueda, Nagano, 386-8567, Japan. rarai@shinshu-u.ac.jp.
¹⁰ Department of Biomolecular Innovation, Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Matsumoto, Nagano, 390-8621, Japan. rarai@shinshu-u.ac.jp.
¹¹ Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, 386-8567, Japan. rarai@shinshu-u.ac.jp.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence / genetics
Molecular Dynamics Simulation
Mutagenesis, Site-Directed / methods
Protein Conformation, alpha-Helical / genetics*
Protein Denaturation
Protein Engineering / methods*
Protein Stability
Protein Structure, Secondary / genetics*
Protein Structure, Secondary / physiology
Proteins / metabolism

Substances

Proteins