The FastDesign protocol in the molecular modeling program Rosetta
iterates between sequence optimization and structure refinement to
stabilize de novo designed protein structures and complexes. FastDesign
has been used previously to design novel protein folds and assemblies
with important applications in research and medicine. To promote
sampling of alternative conformations and sequences, FastDesign includes
stages where the energy landscape is smoothened by reducing repulsive
forces. Here, we discover that this process disfavors larger amino acids
in the protein core because the protein compresses in the early stages
of refinement. By testing alternative ramping strategies for the
repulsive weight, we arrive at a scheme that produces lower energy
designs with more native-like sequence composition in the protein core.
We further validate the protocol by designing and experimentally
characterizing over 4000 proteins and show that the new protocol
produces higher stability proteins.