Gas-phase absorption properties of DsRed model chromophores

Abstract

The absorption spectra of two compounds, RFP(1) and RFP(2), designed to model the chromophore of the Red Fluorescent Protein DsRed have been recorded in the gas phase with a heavy-ion storage ring technique. Both anions and cations were investigated. The electronic delocalization is greater in RFP(2) than in RFP(1) due to an additional CC double bond in conjugation with the π system, and the absorption bands of RFP(2) are red-shifted compared to those of RFP(1). Band maxima of the RFP(2) and RFP(1) anions are 549 nm and 521 nm, respectively, and of the cations 448 nm and 441 nm, respectively. These values are in good agreement with calculated HOMO–LUMO gaps at the B3LYP/6-311++G(2d,p)//PM3 level of theory: 559 nm and 496 nm for the RFP(2) and RFP(1) anions and 452 nm and 436 nm for the corresponding cations. The protein absorbs maximally at 558 nm and it is assumed that the chromophore is anionic. Hence, the electronic structure of the RFP(2) anion is close to that of the in vivo chromophore in its protein environment. A comparison is made between the two model chromophores and their well-known Green Fluorescent Protein homologue chromophore, as well as between different media (gas phase, solution phase). For the anionic gas-phase spectra, vibrational structures are clearly resolved for both compounds (hν₀ = 382 ± 10 cm⁻¹ for RFP(1) and 518 ± 10 cm⁻¹ for RFP(2)) and are assigned to harmonic vibrational progressions due to collective motion of the entire chromophores. Based on calculations on model chromophores closer to the wild-type DsRed chromophore, we suggest that the protein environment forces the chromophore to adopt a planar geometry.