Comparing Stabilization Strategies Between Engineered and Naturally Thermostable Proteins

Abstract

Bioengineers strive to create highly stable and functional proteins for use in biomedical and industrial purposes such as biosensors and pharmaceuticals. In order to better design thermostable proteins de novo, we investigated what features and properties contributed to thermostability in both engineered and naturally occurring thermostable proteins. UMC is a de novo-designed protein that is closely related to the thermostable protein UVF (Tm >99°C), which is an engineered thermostable variant of the Engrailed Homeodomain (EnHD) designed by the Mayo group. The Engrailed Homeodomain (EnHD) is a three-helix bundle transcription factor naturally found in fruit flies. We also studied consensus HD (conHD), a consensus design based on the homeodomain family of proteins that is more thermostable than EnHD. In order to compare stabilization strategies between engineered and naturally thermostable proteins, we compared these proteins to the naturally thermostable protein Cbp2Hb. Cbp2Hb is a six-helix protein found in Hyperthermus butylicus, an archaea that lives in temperatures up to 108 °C. We split the structure of Cbp2Hb into its two three-helix-bundle domains, Cbp2HbN, containing the N-terminal bundle, and Cbp2HbC, containing the C-terminal bundle. We performed all-atom, explicit-solvent molecular dynamic simulations at 25 °C and 100 °C on these four thermostable proteins as well as EnHD and used in lucem molecular mechanics to analyze their dynamics. We analyzed the pairwise residue interactions, solvent accessible surface area, and secondary structures in all five proteins to determine the structural and dynamic basis of the proteins’ thermostability.