Impact of the Protein Environment on Two-Photon Absorption Cross-Sections of the GFP Chromophore Anion Resolved at the XMCQDPT2 Level of Theory

The search for fluorescent proteins with large two-photon absorption (TPA) cross-sections and improved brightness is required their efficient use in bioimaging. Here, we explored the impact of a single-point mutation close to anionic form GFP chromophore on its TPA activity. We considered lowest-energy transition EGFP modification T203I. focused methodology obtaining reliable mutated proteins, based conformational sampling using molecular dynamics simulations high-level XMCQDPT2-based QM/MM approach. also studied numerical convergence sum-over-states formalism provide direct evidence applicability two-level model calculating EGFP. calculated values were found be very sensitive changes permanent dipole moments between ground excited states highly tunable by internal electric field protein environment. In case anion, even single hydrogen bond was shown capable drastically increasing cross-section. Such high tunability nonlinear photophysical properties anions can used rational design brighter bioimaging laser scanning microscopy.