Developing Bright Green Fluorescent Protein (GFP)‐like Fluorogens for Live‐Cell Imaging with Nonpolar Protein−Chromophore Interactions

Abstract

Fluorescence-activating proteins (FAPs) that bind a chromophore and activate its fluorescence have gained popularity in bioimaging. The fluorescence-activating and absorption-shifting tag (FAST) is a light-weight FAP that enables fast reversible fluorogen binding, thus advancing multiplex and super-resolution imaging. However, the rational design of FAST-specific fluorogens with large fluorescence enhancement (FE) remains challenging. Herein, a new fluorogen directly engineered from green fluorescent protein (GFP) chromophore by a unique double-donor-one-acceptor strategy, which exhibits an over 550-fold FE upon FAST binding and a high extinction coefficient of approximately 100,000 M À 1 cm À 1 , is reported. Correlation analysis of the excited state nonradiative decay rates and environmental factors reveal that the large FE is caused by nonpolar proteinÀ fluorogen interactions. Our deep insights into structure-function relationships could guide the rational design of bright fluorogens for live-cell imaging with extended spectral properties such as redder emissions.

A Novel Dialkylamino GFP Chromophore as an Environment-Polarity Sensor Reveals the Role of Twisted Intramolecular Charge Transfer

“…The logarithmic plots of rate constants against the solvent polarity show that log(k FS ) and log(k nr ) are almost linearly correlated with E N T (Figure 3b, top and middle panels) while log(k r ) largely stays constant except for water and EtOAc (Figure 3b, bottom panel; and Table 1, rightmost column). The latter part serves as an intrinsic validation since the radiative transition (fluorescence) lifetime of these GFP-derived chromophores is typically on the few ns timescale [9,26,33,61], in accordance with the current average value of ~8.9 ns (see the dashed line in bottom panel of Figure 3b). In particular, the clear correlation between log(k nr ) and E N T , reminiscent of the Arrhenius equation, suggests that the barrier crossing from FS to TICT is significant with an activation energy strongly dependent on solvent polarity.…”

“…The last green EADS was associated with the decay of FS with an apparent lifetime of 0.78 ns. This assignment was firmly supported by its high resemblance to steady-state fluorescence (the gray-shaded band overlaid in Figure 3a bottom panels) in peak wavelength [26].…”

“…The excited-state electronic spectra of compounds 1, 2, and 3 in various solvents were collected using a home-built femtosecond transient absorption (fs-TA) setup. The detailed description of the optical setup can be found in our previous work [26,29,30]. Excitation wavelengths of 490-505, 500-520, 500-510 nm were used for compound 1, 2, and 3 in various solvents (Figure S1), respectively, with the pump power in the range of 0.2-0.3 mW.…”

“…Quantum calculations by systematic scanning of nuclear coordinates suggested the formation of a TICT state as a result of -NMe 2 rotation to be the main driver for the chromophore fluorogenicity. With femtosecond transient absorption (fs-TA) spectroscopy and a newly developed multivariable analysis approach [26], we quantitatively characterized the intrinsic relationship between the solvent polarity and the solute nonradiative decay rate (mainly due to twisting motion of -NMe 2 reaching a TICT state) and found that the FQY is mainly modulated by H-bonding interactions between the chromophore and solvent molecules.…”

Probing GFP Chromophore Analogs as Anti-HIV Agents Targeting LTR-III G-Quadruplex

“…GFP chromophore and its synthetic analogues find diverse applications in bioimaging and fluorescent probes design [18][19][20][21]. Interestingly, the structural features of these compounds (Figure 1C) resemble, to a significant extent, the second type of G4-binding compounds, the non-fused aromatic systems (Figure 1B).…”