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Title: | Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging | Authors: | Nikolaev, Andrey Yudenko, Anna SMOLENTSEVA, Anastasiia Bogorodskiy, Andrey Tsybrov, Fedor Borshchevskiy, Valentin Bukhalovich, Siarhei V. Nazarenko, Vera Kuznetsova, Elizaveta Semenov, Oleg Remeeva, Alina Gushchin, Ivan |
Issue Date: | 2023 | Publisher: | ELSEVIER | Source: | JOURNAL OF BIOLOGICAL CHEMISTRY, 299 (3) (Art N° 102977) | Abstract: | Flavin-binding fluorescent proteins are promising geneti-cally encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even be-tween different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy. | Notes: | Gushchin, I (corresponding author), Moscow Inst Phys & Technol, Res Ctr Mol Mech Aging & Age Related Dis, Dolgoprudnyi, Russia. ivan.gushchin@phystech.edu |
Keywords: | UV-Vis spectroscopy;flavin;fluorescence;microscopy;protein engineering;Humans;Flavin Mononucleotide;Flavin-Adenine Dinucleotide;HEK293 Cells;Flavoproteins;Riboflavin;Luminescent Proteins | Document URI: | http://hdl.handle.net/1942/40549 | e-ISSN: | 1083-351X | DOI: | 10.1016/j.jbc.2023.102977 | ISI #: | 001007053600001 | Rights: | 2023 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ | Category: | A1 | Type: | Journal Contribution |
Appears in Collections: | Research publications |
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Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging.pdf | Published version | 2.63 MB | Adobe PDF | View/Open |
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