Molecular-Level Insight into the Spectral Tuning Mechanism of the DsRed Chromophore

We present a detailed study of the protein environmental effects on the one- and two-photon absorption (1PA and 2PA, respectively) properties of the <i>S</i>₀-<i>S</i>₁ transition in the DsRed protein using the polarizable embedding density functional theory formalism. We find that steric factors and chromophore–protein interactions act in concert to enhance the 2PA activity inside the protein while adversely blue-shifting the 1PA maximum. A two-state model reveals that the 2PA intensity gain is primarily governed by the increased change in the permanent dipole moment between the ground and the excited states acquired inside the protein. Our results indicate that this mainly is attributable to counter-directional contributions stemming from Lys163 and the conserved Arg95 with the former additionally identified as a key residue in the color tuning mechanism. The results provide new insight into the tuning mechanism of DsRed and suggest a possible strategy for simultaneous improvement of its 1PA and 2PA properties