TY - JOUR
T1 - Rational thermostabilisation of four-helix bundle dimeric de novo proteins
AU - Irumagawa, Shin
AU - Kobayashi, Kaito
AU - Saito, Yutaka
AU - Miyata, Takeshi
AU - Umetsu, Mitsuo
AU - Kameda, Tomoshi
AU - Arai, Ryoichi
N1 - Funding Information:
We thank Prof. Michael Hecht at Princeton University for the kind gift of the expression plasmid of WA20, and Dr. Naoya Kobayashi, Dr. Takahiro Kosugi, Dr. Rie Koga, and Dr. Nobuyasu Koga at the Institute for Molecular Science (IMS) for assistance with the CD experiments. The SAXS and SEC–MALS experiments were performed at Photon Factory (PF), KEK, under the approval of the PF program advisory committee (Proposal Nos. 2016G606 and 2018G634). We thank the beamline scientists and staff at PF, KEK. We are indebted to Research Center for Supports to Advanced Science, Shinshu University for providing facilities. We thank Dr. Tim Cooper from Edanz Group for editing a draft of this manuscript in English. This work was supported by the New Energy and Industrial Technology Development Organization (NEDO) and JSPS KAKENHI Grant Numbers JP16K05841, JP17KK0104, and JP19H02522. This work was also supported by the Joint Research Program of IMS (IMS program No. 214).
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/12
Y1 - 2021/12
N2 - 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 (Tm). 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 (ΔTm = 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 Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.
AB - 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 (Tm). 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 (ΔTm = 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 Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.
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U2 - 10.1038/s41598-021-86952-2
DO - 10.1038/s41598-021-86952-2
M3 - Article
C2 - 33824364
AN - SCOPUS:85103651199
VL - 11
JO - Scientific Reports
JF - Scientific Reports
SN - 2045-2322
IS - 1
M1 - 7526
ER -