RT Journal Article
SR Electronic
T1 Characterization of fluorescent proteins with intramolecular photostabilization
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.03.07.980722
DO 10.1101/2020.03.07.980722
A1 Sarah S. Henrikus
A1 Konstantinos Tassis
A1 Lei Zhang
A1 Jasper H. M. van der Velde
A1 Christian Gebhardt
A1 Andreas Herrmann
A1 Gregor Jung
A1 Thorben Cordes
YR 2020
UL http://biorxiv.org/content/early/2020/03/07/2020.03.07.980722.abstract
AB Genetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro. Since there are no other natural fluorescent tags with comparable features, the impact of fluorescent proteins for biological research cannot be overemphasized. 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. The approach uses direct conjugation of photostablizing compounds such as triplet-state quenchers or redox-active substances to an organic fluorophore, thereby creating high local concentrations of photostabilizer. Here, we introduce an experimental strategy to screen for the effects of covalently-linked photostabilizers on fluorescent proteins. We recombinantly produced a double cysteine mutant (A206C/L221C) of α-GFP for attachment of photostabilizer-maleimides on the ß-barrel in close proximity to the chromophore. Whereas labelling with photostabilizers such as Trolox, Nitrophenyl, 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 of α-GFP to an azobenzene derivative. 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 near-UV and blue spectral range. Our study paves the way towards the development and design of a second generation of fluorescent proteins with photostabilizers placed directly in the protein barrel by methods such as unnatural amino acid incorporation.