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Main Title: Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins
Author(s): Buhrke, David
Tavraz, Neslihan N.
Shcherbakova, Daria M.
Sauthof, Luisa
Moldenhauer, Marcus
Vélazquez Escobar, Francisco
Verkhusha, Vladislav V.
Hildebrandt, Peter
Friedrich, Thomas
Type: Article
Language Code: en
Is Part Of: 10.14279/depositonce-8309
Abstract: Phytochromes are red/far-red light sensing photoreceptors employing linear tetrapyrroles as chromophores, which are covalently bound to a cysteine (Cys) residue in the chromophore-binding domain (CBD, composed of a PAS and a GAF domain). Recently, near-infrared (NIR) fluorescent proteins (FPs) engineered from bacterial phytochromes binding biliverdin IXα (BV), such as the iRFP series, have become invaluable probes for multicolor fluorescence microscopy and in vivo imaging. However, all current NIR FPs suffer from relatively low brightness. Here, by combining biochemical, spectroscopic and resonance Raman (RR) assays, we purified and characterized an iRFP variant that contains a BV chromophore simultaneously bound to two cysteines. This protein with the unusual double-Cys attached BV showed the highest fluorescence quantum yield (FQY) of 16.6% reported for NIR FPs, whereas the initial iRFP appeared to be a mixture of species with a mean FQY of 11.1%. The purified protein was also characterized with 1.3-fold higher extinction coefficient that together with FQY resulted in almost two-fold brighter fluorescence than the original iRFP as isolated. This work shows that the high FQY of iRFPs with two cysteines is a direct consequence of the double attachment. The PAS-Cys, GAF-Cys and double-Cys attachment each entails distinct configurational constraints of the BV adduct, which can be identified by distinct RR spectroscopic features, i.e. the marker band including the C=C stretching coordinate of the ring A-B methine bridge, which was previously identified as being characteristic for rigid chromophore embedment and high FQY. Our findings can be used to rationally engineer iRFP variants with enhanced FQYs.
Issue Date: 12-Feb-2019
Date Available: 18-Feb-2019
DDC Class: 540 Chemie und zugeordnete Wissenschaften
600 Technik
Subject(s): chromophore binding
Sponsor/Funder: DFG, 325093850, Open Access Publizieren 2017 - 2018 / Technische Universität Berlin
BMBF, 01DJ15007, Carotenoidbindende photoschaltbare Proteine: Lichtinduzierte Dynamik und Anwendungen in modernen mikroskopischen Verfahren
DFG, 53182490, EXC 314: Unifying Concepts in Catalysis
DFG, 221545957, SFB 1078: Proteinfunktion durch Protonierungsdynamik
Journal Title: Scientific Reports
Publisher: Nature Publishing Group
Publisher Place: London
Volume: 9
Article Number: 1866
Publisher DOI: 10.1038/s41598-018-38433-2
EISSN: 2045-2322
Appears in Collections:FG Bioenergetik » Publications

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