Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes : fluorescence mechanisms and excited-state dynamics

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Hontani , Y , Shcherbakova , D M , Baloban , M , Zhu , J , Verkhusha , V & Kennis , J T M 2016 , ' Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes : fluorescence mechanisms and excited-state dynamics ' , Scientific Reports , vol. 6 , 37362 . https://doi.org/10.1038/srep37362

Title: Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes : fluorescence mechanisms and excited-state dynamics
Author: Hontani, Yusaku; Shcherbakova, Daria M.; Baloban, Mikhail; Zhu, Jingyi; Verkhusha, Vladislav; Kennis, John T. M.
Other contributor: University of Helsinki, Medicum

Date: 2016-11-18
Language: eng
Number of pages: 12
Belongs to series: Scientific Reports
ISSN: 2045-2322
DOI: https://doi.org/10.1038/srep37362
URI: http://hdl.handle.net/10138/170409
Abstract: Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are of great interest for in vivo imaging. They utilize biliverdin (BV) as a chromophore, which is a heme degradation product, and therefore they are straightforward to use in mammalian tissues. Here, we report on fluorescence properties of NIR FPs with key alterations in their BV binding sites. BphP1-FP, iRFP670 and iRFP682 have Cys residues in both PAS and GAF domains, rather than in the PAS domain alone as in wild-type BphPs. We found that NIR FP variants with Cys in the GAF or with Cys in both PAS and GAF show blue-shifted emission with long fluorescence lifetimes. In contrast, mutants with Cys in the PAS only or no Cys residues at all exhibit red-shifted emission with shorter lifetimes. Combining these results with previous biochemical and BphP1-FP structural data, we conclude that BV adducts bound to Cys in the GAF are the origin of bright blue-shifted fluorescence. We propose that the long fluorescence lifetime follows from (i) a sterically more constrained thioether linkage, leaving less mobility for ring A than in canonical BphPs, and (ii) that pi-electron conjugation does not extend on ring A, making excited-state deactivation less sensitive to ring A mobility.
Subject: CHROMOPHORE-BINDING DOMAIN
IN-VIVO
CRYSTAL-STRUCTURE
CYANOBACTERIUM SYNECHOCYSTIS
UNUSUAL BACTERIOPHYTOCHROME
MIDINFRARED SPECTROSCOPY
SIGNAL-TRANSDUCTION
EXCITATION-ENERGY
QUANTUM YIELD
LIGHT
3111 Biomedicine
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