Local Electric Field Controls Fluorescence Quantum Yield of Red and Far-Red Fluorescent Proteins

Abstract

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.

Characterization of red fluorescent reporters for dual-color in vivo three-photon microscopy

“…The 1P and 2P excitation peaks for tdTomato are 554 and 1050 nm, while mScarlet is maximally excited at 569 nm and has a bimodal 2P excitation curve with peaks at 1065 and 1143 nm ( Fig. 1c ) 28, 29, 30, 31, 32,33 .…”

Energetic basis of excited-state enzyme design and function

“…Two factors mediate excited-state pathway selection: sterics, which acts upon large scale nuclear motion of two rings during isomerization, and electrostatics, which interacts with electronic redistribution during isomerization (or driving force). The electrostatic influence of the red fluorescent protein environment on the corresponding chromophore's FQY is also extensively discussed by a recent paper 42 , while our physical model treats electrostatics differently and explicitly incorporates the steric component (see Section S2 in Supporting Information). According to eq 5, FQY is a nonlinear function of Δ𝜈̅ , and thus the linear additivity of driving force does not translate to an additivity of FQY, as observed from the compensating hybrids (Figure 2D and Table 2).…”

Generation of bright monomeric red fluorescent proteins via computational design of enhanced chromophore packing

“…Although increasing either of these two properties will proportionally increase brightness, it is not well understood how changes to the RFP structure can benecially impact their extinction coefficient, complicating the prediction of benecial mutations by rational design. On the other hand, it is known that the quantum yield of uorophores is directly related to their conformational exibility, [8][9][10] as motions can dissipate the absorbed energy as heat instead of as photons. In the case of uorescent proteins, it has been shown that twisting of the chromophore p-hydroxybenzylidene moiety via torsions in the methine bridge causes non-radiative decay.…”

“…In the case of uorescent proteins, it has been shown that twisting of the chromophore p-hydroxybenzylidene moiety via torsions in the methine bridge causes non-radiative decay. 10,11 Therefore, it should be possible to enhance RFP brightness by designing mutations to restrict the conformational exibility of the phydroxybenzylidene moiety, resulting in higher quantum yield.…”