The ionization states of the protein residues were determined using the PDLD/S-LRA titration method (see section 2.2 for details) for the RLuc8 and its variants. Starting with the high-resolution X-ray structure of reference 11 (PDB ID: 7OMR), we conducted an overall 200 ps relaxation run in the ground state by slowly heating the system from 5 K to 300 K. Subsequently, QCFF/PI MD simulations were performed for 10 ps in the first excited singlet state, with a 0.5 fs time step at 300 K.