Green Fluorescent Protein (GFP) is widely used in bioimaging due to its remarkable fluorescent properties. GFP is also found to undergo oxidative photoconversion, which is accompanied by electron transfer from the excited state of the GFP chromophore anion to oxidants of various origin. Recently, we have shown that light-induced electron transfer from GFP is mediated by a single vibrational mode. The high-frequency in-plane C=C stretching mode facilitates energy exchange between nuclei and electrons on the (sub)picosecond timescale, which is faster than vibrational relaxation. This mode is also active upon photoexcitation, thus resulting in a wavelength-dependent quantum yield of the GFP photoconversion. Here, we aim at enhancing the efficiency of the GFP photoconversion by changing the intensity of the specific vibrational mode, which is coupled to excited-state electron transfer, upon photoexcitation.
We simulate and explore vibronic structures of one-photon (OPA) and two-photon (TPA) absorption profiles of the GFP chromophore anion in the gas phase and inside the protein. The equilibrium geometry parameters, vibrational frequencies, excited-state gradients, and two-photon absorption tensor elements of the GFP chromophore are calculated using time-dependent density functional theory (TDDFT) with the hybrid PBE0/(aug)-cc-pVDZ functional. The ground-state protein calculations are performed using the combined PBE0/CHARMM approach. The parameters used in the TPA two-level model are obtained using the extended multiconfiguration quasi-degenerate perturbation theory (XMCQDPT2), coupled to the effective fragment potential method for treating the protein environment in the excited-state protein calculations. The OPA and TPA vibrational profiles are obtained using a linear coupling scheme, within the double harmonic parallel-mode approximation, accounting for both Franck-Condon and Herzberg-Teller couplings.
We show that the TPA and OPA spectral profiles are markedly different. The TPA maximum is blue shifted compared to that observed in the OPA spectrum, coinciding with the 0-1 excitation of the C=C stretching mode. Furthermore, by using a two-level model we show that the TPA cross-section and the contribution from each vibrational mode can be estimated using simple parameters, such as a difference between permanent dipole moments in the ground and excited states and a transition dipole moment, as well as their derivatives with respect to normal mode coordinates. These parameters can be readily evaluated at a high level of theory. Importantly, we find that the major contribution to the TPA cross-section comes from those vibrational modes that modulate permanent dipole moments. These are the infrared active modes. We therefore conclude that the intensity of the IR-active modes, including the specific mode coupled to light-induced electron transfer from GFP, can be significantly enhanced when switching from conventional one-photon excitation to a non-linear two-photon absorption regime. Our findings pave the way for TPA-enhanced wavelength-dependent photoconversion of GFP.
This work is supported by The Russian Science Foundation (grant no. 17-13-01276). The research is carried out using the equipment of the shared research facilities of HPC computing resources at Lomonosov Moscow State University as well as the local resources provided through the Lomonosov Moscow State University Program of Development.