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. 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.

Self-Assembling Lectin Nano-Block Oligomers Enhance Binding Avidity to Glycans

“…Lectin nano-blocks were designed by fusing the dimeric de novo protein WA20 to the dimeric lectin ACG ( Figure 1 ). The WA20_H86K mutant was used as a component of the lectin nano-block in this study as it was a thermally stabilized mutant with a 3.5 °C higher denaturation midpoint temperature ( T m ) than the original WA20 [ 11 ] and formed a stable dimer in solution ( Figure S1A ). Thereafter, “WA20” refers to the WA20_H86K mutant in this study.…”

“…Moreover, based on the rational prediction of stabilizing mutations using high-temperature molecular dynamics (MD) simulations, three mutations (N22A, N22E, and H86K) of WA20 were found. A double mutant (N22E and H86K) of SUWA (rationally optimized SUWA, ROSA) showed the highest T m (129.0 °C) [ 11 ].…”

Comprehensive mutagenesis to identify amino acid residues contributing to the difference in thermostability between two originally thermostable ancestral proteins

“…Several methods for creating more thermostable proteins have been proposed. Two conventional methods to create proteins with enhanced thermostability are rational design [ 9 , 14 , 26 , 27 ] and directed evolution [ 3 , 28 – 30 ]. As alternative approaches, consensus design and ancestral sequence reconstruction (ASR) have also been developed to create thermostable proteins.…”

“…More stable proteins are preferred as potential scaffolds for protein engineering [ 49 , 50 ]. Therefore, it is an attractive challenge to further improve the thermostability of an originally thermostable protein [ 9 , 51 ]. The two ancestral NDKs, Bac1 and Arc1, were created in our previous ASR experiments [ 22 ].…”

“…Comparative structural analyses between thermophilic and mesophilic protein structures have provided insights into the molecular mechanisms responsible for the thermostability of thermophilic proteins [ 5 , 6 ]. The mechanisms suggested by those studies include the formation of intra- or inter-molecular ion-pairs and ion-pair networks in the native structure [ 7 – 9 ] and also in the denatured state [ 10 ], increased hydrophobicity and improved packing in the hydrophobic core [ 11 – 14 ], shorter loops [ 15 , 16 ], improved subunit-subunit interactions [ 3 , 17 , 18 ] and entropic advantages due to the increased flexibility of native structures [ 19 ]. However, we have yet to reach a comprehensive understanding of the mechanism of protein thermostability [ 20 ].…”