Simulation of the UV/Visible Absorption Spectra of Fluorescent Protein Chromophore Models

Time-dependent density functional theory has been used to simulate the UV/Vis absorption spectra of fluorescent protein model chromophores with particular focus on the vibronic structure of the lowest-energy absorption band as a structural fingerprint. Combining the B3LYP-35 XC functional with non-equilibrium solvation and the gradient approach provides a vibronic structure close to that recorded experimentally (when comparisons with experiment are possible). This study focuses on the chromophore present in green fluorescent proteins (FP1), in its neutral and deprotonated forms as well as for both cis (Z) and trans (E) conformations. The method was then employed to simulate the spectra of structures for which experimental spectra are not available or not well resolved. The effect of extending the conjugation of the chromophore, by adding unsaturated moieties such as in the chromophore of DsRed (FP2), was considered. Overall, among these three investigated structural modifications (neutral versus anionic, smaller versus larger chromophore, and cis versus trans conformations), absorption spectra can only distinguish unambiguously between the neutral and anionic forms. An extension of the π-conjugation length of the chromophore is, as would be expected, reflected in a bathochromic shift of the absorption band maximum, but modifications of the chromophore environment in proteins can also induce shifts of the absorption maximum, and they are difficult to distinguish from each other.