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 (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.
Highlights
The stability of proteins is an important factor for industrial and medical applications
We succeeded in dramatically improving the stability of WA20 by introducing five amino acid substitutions (H26L, G28S, N34L, V71L, and E78L)[20] to enhance the hydrophobic core and α-helix stability based on the WA20 structure
To select target residues for mutations, we searched for hydrophilic residues buried in the WA20 protein structure based on the accessible surface area (ASA) per residue (Supplementary Table S1)
Summary
The stability of proteins is an important factor for industrial and medical applications. 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 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). With the long-term goal of producing nanostructures with extremely high stabilities for applications in nanotechnology, we were motivated to stabilise the designed protein WA20, a main component of PN-Blocks. We succeeded in dramatically improving the stability of WA20 by introducing five amino acid substitutions (H26L, G28S, N34L, V71L, and E78L)[20] to enhance the hydrophobic core and α-helix stability based on the WA20 structure This mutant, which is called super WA20 (SUWA), showed an extremely high denaturation midpoint temperature (Tm) above the boiling point of water
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