Understanding the balance between thermostability and function in engineered proteins is critical for rational protein design. Engineering proteins that are both thermostable and functional present a challenge, as protein function is tightly linked to structure and thermodynamics. We assess the tradeoffs between designing for thermostability versus function by building chimeras designed from a mesostable DNA-binding protein and a thermostable, non-functional homolog. The engrailed homeodomain (EnHD) is a well-characterized DNA-binding transcription factor from D. melanogaster with a moderate thermal stability (T m = 52°C), making it a useful template for protein design. UVF, an engineered version of EnHD, has exceptional thermostability (T m >99°C), which we hypothesize is due in part to its fully hydrophobic core, but it lacks the ability to bind DNA. To investigate whether UVF’s fully hydrophobic core could impart thermostability without destroying EnHD’s DNA-binding function, we designed chimeras incorporating DNA-binding residues from EnHD and the hydrophobic core of UVF. Using molecular dynamics (MD) simulations, we evaluated the proteins’ thermostability by analyzing backbone root mean square deviation (RMSD), hydrophobic core residue-residue contacts, and solvent accessible surface area (SASA) in simulations at 25°C and 100°C. In parallel, we examined DNA-binding function by comparing MD simulations of the protein-DNA complexes, quantifying binding stability through intermolecular distances. To complement our theoretical work, we expressed and purified EnHD, UVF, and the most promising of the chimeras. We assessed DNA-binding function with electrophoretic mobility shift assays and thermostability with temperature melts monitored by circular dichroism. Our findings provide insight to the interplay between thermostability and intermolecular interactions in protein engineering.
Tani et al. (2026) studied this question.