SmURFP

Small ultra red fluorescent protein (smURFP) is a class of far-red fluorescent protein developed through directed evolution 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 outperforms mCherry and tdTomato in living cells. Single-molecule smURFPs emit twice as many photons before photobleaching than small-molecule dyes AlexaFluor647 and Cyanine5. 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. smURFP has the largest two-photon cross-section measured for a fluorescent protein. There are two peak cross-sections of 1,060 and 60 GM at 820 and 1,196 nm, respectively. 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. The crystal structure of the smURFP (PDB: 7UQA) was determined and used to understand the directed evolution. smURFP was also compared to the parental α-allophycocyanin. The crystal structure of a smURFP mutant (PDB: 6FZN​) was published by Fuenzalida-Werner et al. The mutants show significantly larger chromophore pockets and protein volume, which results in diminished quantum yield. 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 and nuclear membrane of mammalian cells.

Chromophore availability in cells and mice

Despite showing comparable biophysical brightness to eGFP when purified protein was normalized, this was not seen in living cells. This suggested there was not enough chromophore (biliverdin) within cells. Addition of biliverdin increased fluorescence, but smURFP with biliverdin was not comparable to eGFP. Biliverdin has two carboxylates at neutral pH and this is inhibiting cellular entry. Biliverdin dimethyl ester is a more hydrophobic analog and readily crosses the cellular membrane. smURFP with biliverdin dimethyl ester shows comparable fluorescence to eGFP in cells and is brighter than bacterial phytochrome fluorescent proteins. The free chromophore can be differentiated from chromophore attached to smURFP by fluorescence lifetime imaging (FLIM) in living cells. Free biliverdin dimethyl ester (BVMe2) has a fluorescence lifetime of 0.586 ns, while BVMe2 attached to smURFP has a fluorescence lifetime of 1.27 ns.

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