A Dynamic Hydrophobic Core and Surface Salt Bridges Thermostabilize a Designed Three-Helix Bundle

Abstract

Thermostable proteins are advantageous in industrial applications, as pharmaceuticals or biosensors, and as templates for directed evolution. As protein-design methodologies improve, bioengineers are able to design proteins to perform a desired function. Although many rationally designed proteins end up being thermostable, how to intentionally design de novo, thermostable proteins is less clear. UVF is a de novo-designed protein based on the backbone structure of the Engrailed homeodomain (EnHD) and is highly thermostable (T m > 99°C vs. 52°C for EnHD). Although most proteins generally have polar amino acids on their surfaces and hydrophobic amino acids buried in their cores, protein engineers followed this rule exactly when designing UVF. To investigate the contributions of the fully hydrophobic core versus the fully polar surface to UVF’s thermostability, we built two hybrid, chimeric proteins combining the sets of buried and surface residues from UVF and EnHD. Here, we determined a structural, dynamic, and thermodynamic explanation for UVF’s thermostability by performing 4 μ s of all-atom, explicit-solvent molecular dynamics simulations at 25 and 100°C, Tanford-Kirkwood solvent accessibility Monte Carlo electrostatic calculations, and a thermodynamic analysis of 40 temperature runs by the weighted-histogram analysis method of heavy-atom, structure-based models of UVF, EnHD, and both chimeric proteins. Our models showed that UVF was highly dynamic because of its fully hydrophobic core, leading to a smaller loss of entropy upon folding. The charged residues on its surface made favorable electrostatic interactions that contributed enthalpically to its thermostability. In the chimeric proteins, both the hydrophobic core and charged surface independently imparted thermostability.

pH and Charged Mutations Modulate Cold Shock Protein Folding and Stability: A Constant pH Monte Carlo Study

“…Red bars mean that the residue has a positive Δ G qq and its side chain is highly exposed to the solvent (solvent-accessible surface area (SASA) > 50%). For these residues, mutations with opposite charge tend to stabilize the native state of the protein, whereas the change of residues with strongly negative Δ G qq usually promotes a destabilization. , …”

“…For these residues, mutations with opposite charge tend to stabilize the native state of the protein, whereas the change of residues with strongly negative ΔG qq usually promotes a destabilization. 17,43 The Bs-CspB WT (PDB ID 1CSP) presents a ΔG elec (sum of all ΔG qq ) of −17.16 kJ/mol. The mutations A46K and S48R present in Bs-CspB variant (Figure 1) promote a decrease of 11.15 kJ/mol, resulting in a ΔG elec = −28.31 kJ/mol.…”

“…The all-atoms coordinate of the native structure of the protein is used as an input of this program, and the Metropolis Monte Carlo algorithm samples the protonation states of ionizable atoms. The server provides quantitative information about the importance of each residue to protein thermal stability, and it has been a robust tool to find rational mutations that improve thermal stability in different proteins. ,, This tool was used to select a specific residue to mutate and make the Bs-CspB variant even more thermostable.…”

“…The server provides quantitative information about the importance of each residue to protein thermal stability, and it has been a robust tool to find rational mutations that improve thermal stability in different proteins. 17,41,43 This tool was used to select a specific residue to mutate and make the Bs-CspB variant even more thermostable.…”

Recent Progress Using De Novo Design to Study Protein Structure, Design and Binding Interactions

“…In 2019 Catrina Nguyen et al investigated the contributions of a fully hydrophobic core and hydrophilic surface to a protein’s thermostability [ 64 ]. This was done by creating two hybrid chimera proteins which combined the buried fully hydrophobic core residues and polar surface residues of UVF with the surface and core residues of EnHD (hybrid one had UVF’s core and EnHD’s surface and the second hybrid was vice versa).…”