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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 −1 cm −1 , is reported. Correlation analysis of the excited state nonradiative decay rates and environmental factors reveal that the large FE is caused by nonpolar protein−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.
Chen et al. (2021) studied this question.