Rational thermostabilisation of four-helix bundle dimeric de novo proteins

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.

Table S1 (continued). Accessible surface areas (ASA) of amino acid residues in the WA20 structure.
The data (chain B) of the crystal structure of WA20 (PDB ID: 3VJF) 16 were calculated by the program AREAIMOL 22,23 in the CCP4 suite 24 . Red letters represent hydrophilic residues with small ASA ratio (ASA ratio ≤ 0.11). (ASA ratio: ratio of ASA to calculated GXG (Gly-Xaa-Gly) value.) The target residues for mutations selected in this study are shown in bold letters.  Figure S1. Potential target residues on the interface of α-helices in the WA20 structure. Two target residues (N22 and N34) on the interface of α-helices were chosen to potentially enhance helix-helix interactions. The target residues are shown as sticks. Chains A and B of the crystal structure of WA20 (PDB: 3VJF) 16 are shown in magenta and cyan, respectively.  MD simulations under high temperature condition (600 K, 10 ns) were performed for WA20 and the thermostabilised mutants reported in a previous study 20 . Root mean square deviations (RMSD) between initial structures and structures after high-temperature MD simulations were calculated to evaluate the degrees of protein unfolding. MD simulations were performed ten times for each protein. There is a strong negative correlation between RMSD and experimentally determined T m of WA20 and the mutants (r = −0.966, p = 5.461 × 10 −5 ). MD simulations under high temperature condition (600 K, 10 ns) were performed for WA20 and all of the possible single mutants of WA20 generated by in silico saturation mutagenesis at the four target residue sites. Root mean square deviations (RMSDs) between the initial structures and the structures after hightemperature MD simulations were calculated to evaluate the degrees of protein unfolding. MD simulations were performed ten times for each mutant. Blue bars represent the original WA20. Red bars of small RSMD values represent the mutants selected for experimental evaluations in this study.

Figure S4. SDS-PAGE analysis of WA20 and the WA20 mutants.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the WA20 mutants were performed for the eluted fractions by immobilised metal ion affinity chromatography (IMAC) with TALON metal affinity resin (Takara Bio, Otsu, Japan). The protein bands surrounded in the black square show purified WA20 and the mutants (molecular mass of a monomer of WA20 and its mutants: ~12.5 kDa). Protein molecular weight markers (  The Y-axis represents the normalised value of [θ] 222 nm . The experimental data were fitted to a twostate model with ΔC p fixed to zero using CDpal 33 . The T m values of the WA20 mutants and the SUWA mutants are shown in Table 1 and Table 2, respectively. The graphs were created using CDpal 33 , version 2.18 (https://github.com/PINT-NMR/CDpal/).

Figure S9. Size exclusion chromatography (SEC) analyses of ROWA and ROSA dimers.
SEC profiles of IMAC-purified samples of ROWA (left) and ROSA (right) with a Superdex 75 increase 10/300 GL column (Cytiva) are shown in top panels. The isolated fractions of the dimer peaks of ROWA and ROSA were reanalysed immediately after the SEC purification (0 day, middle panels) and after keeping them at 20 ℃ for 7 days (bottom panels). These results suggest that the dimers did not change to the other oligomers, i.e., the oligomeric states do not exchange one another on a timescale of a week. The Y-axis represents the intensity of UV absorbance (A 280 nm ).
13 Figure S11. Plots of the scattering curves calculated from the DAMMIN models fitting to the experimental SAXS data.
The concentration-normalised SAXS intensity, I(q)/c, of the ROWA and ROSA samples (black open circle) and that optimised by the DAMMIN procedure (red line). The χ 2 value represents the degree of fitting between the experimental data and the data calculated from the DAMMIN model.  The left panels show the structure of WA20. The middle and right panels show the model structures of the N22K and N22Lmutants, respectively. Chains A and B of WA20 are shown in magenta and cyan, respectively. The target and mutated residues are shown as green sticks in the upper panels. In the lower panels, all atoms including hydrogens are shown as spheres, and colors are the same as the upper panels. The model structures were constructed from the crystal structure of WA20 (PDB ID: 3VJF) and optimised by MD simulation at 300 K for 1 ns.