Genetically encoded probes with red-shifted absorption and fluorescence are highly desirable for imaging applications because they can report from deeper tissue layers with lower background and because they provide additional colors for multicolor imaging. Unfortunately, red and especially far-red fluorescent proteins have very low quantum yields, which undermines their other advantages. Elucidating the mechanism of nonradiative relaxation in red fluorescent proteins (RFPs) could help developing ones with higher quantum yields. Here we consider two possible mechanisms of fast nonradiative relaxation of electronic excitation in RFPs. The first, known as the energy gap law, predicts a steep exponential drop of fluorescence quantum yield with a systematic red shift of fluorescence frequency. In this case the relaxation of excitation occurs in the chromophore without any significant changes of its geometry. The second mechanism is related to a twisted intramolecular charge transfer in the excited state, followed by an ultrafast internal conversion. The chromophore twisting can strongly depend on the local electric field because the field can affect the activation energy. We present a spectroscopic method of evaluating local electric fields experienced by the chromophore in the protein environment. The method is based on linear and two-photon absorption spectroscopy, as well as on quantum-mechanically calculated parameters of the isolated chromophore. Using this method, which is substantiated by our molecular dynamics simulations, we obtain the components of electric field in the chromophore plane for seven different RFPs with the same chromophore structure. We find that in five of these RFPs, the nonradiative relaxation rate increases with the strength of the field along the chromophore axis directed from the center of imidazolinone ring to the center of phenolate ring. Furthermore, this rate depends on the corresponding electrostatic energy change (calculated from the known fields and charge displacements), in quantitative agreement with the Marcus theory of charge transfer. This result supports the dominant role of the twisted intramolecular charge transfer mechanism over the energy gap law for most of the studied RFPs. It provides important guidelines of how to shift the absorption wavelength of an RFP to the red, while keeping its brightness reasonably high.
Tetraphenylethenyl-Modified 1,8-Naphthalimide Dye with Efficient Aggregation-Enhanced Emisssion, Solvatochromism and Intramolecular Charge Transfer Characteristics
