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

Yusaku Hontani,Jingyi Zhu,John T M Kennis,Mikhail Baloban,Vladislav V Verkhusha,Daria M Shcherbakova

doi:10.1038/srep37362

Yusaku Hontani, Jingyi Zhu + Show 4 more

Open Access

https://doi.org/10.1038/srep37362

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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 π-electron conjugation does not extend on ring A, making excited-state deactivation less sensitive to ring A mobility.

Highlights

Near-infrared (NIR) fluorescent proteins (FPs) are of great interest for in vivo imaging of mammals
These Cys residues in the GAF domain of BphP1-FP, iRFP670 and iRFP682 were introduced at the same position as the conserved Cys residues in plant and cyanobacterial phytochromes, which bind phycocyanobilin (PCB) or phytochromobilin (Pφ, capical B)[7] (Fig. S1)
The substantially prolonged lifetimes and enhancement of fluorescence intensity were identified in the blue-shifted Near-infrared fluorescent proteins (NIR FPs) variants, while the isomerization processes were found to be completely blocked

Summary

Introduction

Near-infrared (NIR) fluorescent proteins (FPs) are of great interest for in vivo imaging of mammals. Several blue-shifted bright NIR FPs have been engineered from wild-type RpBphP1, RpBphP2, and RpBphP615, such as BphP1-FP (the brightest FP among current BphP-derived NIR FPs)[16], iRFP68214 and iRFP67014, respectively. In these NIR FPs, the PHY domain was truncated, and photoisomerization was blocked through mutation of key residues in the GAF domain. A Cys residue was introduced in the GAF domain: Cys[253] in BphP1-FP, Cys[244] in iRFP670 and Cys[249] in iRFP682, enabling BV to covalently bind to cysteine residues in the PAS and GAF domains[14,15]. Real-time studies of the picosecond dynamics on the excited states are required to understand molecular mechanisms of fluorescence more deeply

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