Ultrafast photophysics of the cyan fluorescent protein chromophore in solution

Incorporation of fluorescent proteins (FPs) into biological systems has revolutionised bioimaging and the understanding of cellular processes. Ongoing developments of FPs are driving efforts to characterise the fundamental photoactive unit (chromophore) embedded within the protein. Cyan FP has a blue emitting chromophore and is widely used in Förster resonance energy transfer studies. Here, we probe the ultrafast photophysics of the cyan FP chromophore in solution using time-resolved fluorescence up-conversion and transient absorption spectroscopies. The ultrafast dynamics are characterised by two lifetimes, sub-picosecond <i>τ</i>₁ (or <i>τ</i>_F) associated with loss of the fluorescent Franck-Condon state, and lifetime <i>τ</i>₂ on the order of several picoseconds that is linked with cooling of a hot ground state. MRSF-TDDFT calculations show that the relaxed S₁ state equilibrium geometry is classified as a partial twisted intramolecular charge-transfer state, and lies close in energy to a conical intersection seam associated with torsion about the central double bond leading to facile internal conversion. The excited state dynamics exhibit only a weak viscosity dependence, consistent with a barrierless and near-volume-conserving non-radiative decay mechanism. Fluorescence lifetimes for the deprotonated anion are twice those for the neutral.