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Nat Commun. 2019 Jan 17;10(1):279. doi: 10.1038/s41467-018-08050-8.

Smallest near-infrared fluorescent protein evolved from cyanobacteriochrome as versatile tag for spectral multiplexing.

Author information

1
Medicum, Faculty of Medicine, University of Helsinki, 00290, Helsinki, Finland.
2
Department of Anatomy and Structural Biology, and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, 10461, USA.
3
Basic Science Program, Macromolecular Crystallography Laboratory, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, MD, 21702, USA.
4
Medicum, Faculty of Medicine, University of Helsinki, 00290, Helsinki, Finland. vladislav.verkhusha@einstein.yu.edu.
5
Department of Anatomy and Structural Biology, and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, 10461, USA. vladislav.verkhusha@einstein.yu.edu.

Abstract

From a single domain of cyanobacteriochrome (CBCR) we developed a near-infrared (NIR) fluorescent protein (FP), termed miRFP670nano, with excitation at 645 nm and emission at 670 nm. This is the first CBCR-derived NIR FP evolved to efficiently bind endogenous biliverdin chromophore and brightly fluoresce in mammalian cells. miRFP670nano is a monomer with molecular weight of 17 kDa that is 2-fold smaller than bacterial phytochrome (BphP)-based NIR FPs and 1.6-fold smaller than GFP-like FPs. Crystal structure of the CBCR-based NIR FP with biliverdin reveals a molecular basis of its spectral and biochemical properties. Unlike BphP-derived NIR FPs, miRFP670nano is highly stable to denaturation and degradation and can be used as an internal protein tag. miRFP670nano is an effective FRET donor for red-shifted NIR FPs, enabling engineering NIR FRET biosensors spectrally compatible with GFP-like FPs and blue-green optogenetic tools. miRFP670nano unlocks a new source of diverse CBCR templates for NIR FPs.

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