Developing Bright GFP-Like Fluorogens for Live-Cell Imaging with Nonpolar Protein-Chromophore Interactions

• Published in: Chemistry : a European journal
• Main Authors: Chen, Cheng, Tachibana, Sean R, Baleeva, Nadezhda S, Myasnyanko, Ivan N, Bogdanov, Alexey M, Gavrikov, Alexey S, Mishin, Alexander S, Malyshevskaya, Kseniya K, Baranov, Mikhail S, Fang, Chong
• Format: Journal Article
• Language: English
• Published: Germany 02-05-2021
• Subjects: multivariable analysis; yellow fluorogen design; femtosecond transient absorption; fluorescence enhancement; biomimetics
• ISSN: 1521-3765

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. Here, we report a novel 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 ~100,000 M -1 ·cm -1 . 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.