Characterization of Fluorescent Proteins with Intramolecular Photostabilization. (22nd July 2021)

• Record Type: Journal Article
• Authors: Henrikus, Sarah S.; Tassis, Konstantinos; Zhang, Lei; van der Velde, Jasper H. M.; Gebhardt, Christian; Herrmann, Andreas; Jung, Gregor; Cordes, Thorben
• Is Part Of: Chembiochem. Volume 22:Number 23(2021)
• Page Start: 3283
• Page End: 3291
• Publication Date: 2021-07-22
• Subjects: fluorescent proteins -- spectroscopy -- photophysics -- photostabilization -- self-healing dyes; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.202100276
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: Genetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro . Despite their importance, their photophysical properties, i. e., brightness, count‐rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties of organic fluorophores. Here, direct conjugation of triplet‐state quenchers or redox‐active substances creates high local concentrations of photostabilizer around the fluorophore. In this paper, we screen for effects of covalently linked photostabilizers on fluorescent proteins. We produced a double cysteine mutant (A206C/L221C) of α‐GFP for attachment of photostabilizer‐maleimides on the β‐barrel near the chromophore. Whereas labelling with photostabilizers such as trolox, a nitrophenyl group, and cyclooctatetraene, which are often used for organic fluorophores, had no effect on α‐GFP‐photostability, a substantial increase of photostability was found upon conjugation to azobenzene. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet‐energy of azobenzene might be crucial for triplet‐quenching of fluorophores in the blue spectral range. Our study paves the way for the development of fluorescent proteins with photostabilizers in the protein barrel by methods such as unnatural amino acid incorporation. Abstract : An experimental strategy is introduced to characterize the effects of covalently linked photostabilizers on fluorescent proteins. α‐GFP variant A206C/L221C allowed attachment of photostabilizer‐maleimides (trolox, nitrophenyl, cyclooctatetraene and azobenzene) on the β‐barrel in proximity to the chromophore. Increases of photostability were found upon conjugation of azobenzene with a photostability mechanism to be elucidated.