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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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3T3 Cells
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Animals
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Bacterial Proteins / chemistry*
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Bacterial Proteins / genetics
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Bacterial Proteins / isolation & purification
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Bacterial Proteins / metabolism
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Biliverdine / metabolism
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Biosensing Techniques / methods*
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Cyanobacteria / chemistry*
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Cyanobacteria / genetics
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Cyanobacteria / metabolism
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Directed Molecular Evolution / methods
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Female
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Fluorescence
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HeLa Cells
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Humans
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Intravital Microscopy / methods
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Luminescent Proteins / chemistry*
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Luminescent Proteins / genetics
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Luminescent Proteins / isolation & purification
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Luminescent Proteins / metabolism
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Mice
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Microscopy, Fluorescence / methods
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Mutagenesis
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Neurons
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Optogenetics / methods
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Photoreceptors, Microbial / chemistry*
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Photoreceptors, Microbial / genetics
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Photoreceptors, Microbial / isolation & purification
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Photoreceptors, Microbial / metabolism
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Primary Cell Culture
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Protein Domains / genetics
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Protein Engineering
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Rats
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Spectroscopy, Near-Infrared / methods
Substances
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Bacterial Proteins
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Luminescent Proteins
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Photoreceptors, Microbial
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Recombinant Proteins
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Biliverdine