Gene

Autofluorescence

Neuroscience

Biology

Förster resonance energy transfer

Photochemistry

Materials science

Protein engineering

Phytochrome

Heme

Biochemistry

Botany

Biliverdin

Molecular engineering

Physics

Nanotechnology

Organic chemistry

Optics

Green fluorescent protein

Near-infrared spectroscopy

Enzyme

Fluorescence

Chromophore

Chemistry

Heme oxygenase

Fluorescent protein

Biophysics

Red light

Nature Communications

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.

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