The Trade-off between Thermostability and Function in Designed DNA-Binding Proteins

Abstract

Protein designers are ever improving their ability to design proteins that fold stably and perform a function, yet meeting both of these goals at once has been a challenge. Here we investigate whether there is a trade-off between thermostability and function by designing chimeras of two three-helix bundle proteins. The Engrailed homeodomain is a DNA-binding protein that serves as a transcription factor in D. melanogaster development and has a melting temperature of 52°C. Based on its backbone structure, a variant, UVF, was previously designed and found to be thermostable (Tm > 99°C) but was not functional. Molecular dynamics and coarse-grained simulations of chimeras of the two proteins suggest that UVF's fully hydrophobic core contributed entropically to its stability, and increased salt bridges on its surface contributed enthalpically. Here, we used equilibrium unfolding to test these thermodynamic predictions. Further, we used molecular dynamics simulations of the proteins bound to DNA to design and assess mutations to the chimeras to improve upon their DNA binding function. DNA-binding was then tested experimentally using electrophoretic mobility shift assays.