Alternative Computational Protocols for Supercharging Protein Surfaces for Reversible Unfolding and Retention of Stability
Both protocols begin by defining the surface of the protein of interest, and if provided, reading a residue file that specifies residues to not mutate. AvNAPSA forcibly mutates NQ and DE/RK in order of solvent accessibility. Rosetta uses Monte Carlo side chain placement guided by computed energies to mutate any surface residue except G, P, C, and hydrogen-bonded side chains, and charged-residue reference energies are adjusted to vary net charge. Both protocols are set up to achieve a desired net charge (above), or to specified reference energies (Rosetta) or surface cutoff (AvNAPSA). Output includes the PDB coordinate file of the redesigned protein, the residue file specifying the allowed mutations, and a log file with information such as residues mutated, number of mutations, net charge, residue energies, and a PyMOL selection command to conveniently view the mutations.