SmURFP

Small ultra red fluorescent protein (smURFP) is a class of far-red fluorescent protein evolved from a cyanobacterial ('' Trichodesmium erythraeum'') phycobiliprotein, α-allophycocyanin. Native α-allophycocyanin requires an exogenous protein, known as a lyase, to attach the chromophore, phycocyanobilin. Phycocyanobilin is not present in mammalian cells. smURFP was evolved to covalently attach phycocyanobilin without a lyase and fluoresce, covalently attach biliverdin (ubiquitous to mammalian cells) and fluoresce, blue-shift fluorescence to match the organic fluorophore, Cy5, and not inhibit ''E. coli'' growth. smURFP was found after 12 rounds of random mutagenesis and manually screening 10,000,000 bacterial colonies.

Properties

smURFP is a homodimer with absorption and emission maximum of 642 nm and 670 nm, respectively. A tandem dimer smURFP (TDsmURFP) was created and has similar properties to smURFP. smURFP is extremely stable with a protein degradation half-life of 17 hour and 33 hour without and with chromophore (biliverdin), respectively. This is comparable to the jellyfish-derived enhanced green fluorescent protein (eGFP) protein degradation half-life of 24 hour. smURFP is extremely photostable and out performs mCherry and tdTomato in living cells. The extinction coefficient (180,000 M⁻¹ cm⁻¹) of smURFP is extremely large and has a modest quantum yield (0.18), which makes it comparable biophysical brightness to eGFP and ~2-fold brighter than most red or far-red fluorescent proteins derived from coral. Despite being a homodimer, all tested N- and C- terminal fusions show correct cellular localization, including the difficult fusion to α-tubulin and Lamin B1 (Figure). smURFP is named after the Smurfs, due to its light blue appearance in white light.

An Excel sheet of the smURFP absorbance, excitation, and emission spectra can be downloade
here
The crystal structure of a smURFP mutant () was published i
Fuenzalida-Werner et al
A 2020 review discusses recent applications of smURFP as a genetically encoded or exogenous probe for ''in vivo'' imaging and discusses problems with biliverdin availability.

smURFP used as nanoparticles, exogenous probes, and ''in vitro'' assays

Free smURFP is 2-3 nm in diameter. smURFP nanoparticles of ~10-14 nm diameter can be synthesized in an oil and water emulsion and remain fluorescent. These fluorescent protein nanoparticles are stable in living mice and useful for non-invasive tumor fluorescence imaging.

Purified smURFP survives ultrasound and fixation to allow fluorescence imaging of macromolecule delivery by ultrasound into corneas.

Free smURFP, purified protein and not genetically encoded, can be encapsulated into viruses and used for non-invasive, fluorescence imaging of biodistribution in living mice.

smURFP covalently attaches biliverdin to turn on fluorescence and is inherently a biliverdin sensor. Researchers showed purified smURFP has a limit of detection of 0.4 nM for biliverdin in human serum. smURFP allows for the creation of ''in vitro'' assays to detect enzyme activity. An assay was developed for thrombin with a detection range of 1.07 aM–0.01 mM and a limit of detection of 0.2 aM.

Tandem dimer smURFP (TDsmURFP) was used as an exogenous fluorescent marker to label the seven-transmembrane receptor Smoothened ( SMO). TDsmURFP was purified from ''E. coli'' and attached to SMO by sortase-mediated conjugation for fluorescence-activated cell sorting (FACS). This novel, exogenous fluorescent protein labeling avoids screening multiple protein insertion sites, organic solvents, and chemical reactions that misfold, inactivate, or degrade proteins.

smURFP is a self-labeling protein

The small Ultra-Red Fluorescent Protein (smURFP) is a self-labeling protein like Halo-, SNAP-, and CLIP-tags. The smURFP-tag accepts a biliverdin substrate modified on a carboxylate with a polyethylene glycol (PEG) linker to the cargo molecule. Unlike the Halo-, SNAP-, and CLIP-tags that use the substrate to only covalently attach the cargo molecule, biliverdin is fluorogenic, and fluorescence is turned "on" with covalent attachment to the smURFP-tag to allow far-red fluorescence tracking of cargo molecule in living cells. Biliverdin also quenches fluorescein cargo to allow for imaging without substrate removal. Biliverdin modification on a single carboxylate creates a neutral molecule that passes the outer