Molecular dynamics simulations suggest stabilizing mutations in a de novo designed α/β protein

Designing functional proteins that can withstand extreme heat is beneficial for industrial and protein therapeutic applications. Thus, elucidating the atomic-level determinants of thermostability is a major interest for rational protein design. To that end, we compared the structure and dynamics of a set of previously designed, thermostable proteins based on the activation domain of human procarboxypeptidase A2 (AYEwt). The mutations in these designed proteins were intended to increase hydrophobic core packing and inter-secondary-structure interactions. To evaluate whether these design strategies were successfully deployed, we performed all-atom, explicit-solvent molecular dynamics (MD) simulations of AYEwt and three designed variants at both 25 and 100°C. Our MD simulations agreed with the relative experimental stabilities of the designs based on their secondary structure content, Cα root-mean-square deviation/fluctuation, and buried-residue solvent accessible surface area. Using a contact analysis, we found that the designs stabilize inter-secondary structure interactions and buried hydrophobic surface area, as intended. Based on our analysis, we designed three additional variants to test the role of helix stabilization, core packing, and a Phe → Met mutation on thermostability. We performed the additional MD simulations and analysis on these variants, and these data supported our predictions.