# SmURFP
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> Source: https://en.wikipedia.org/wiki/SmURFP
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thumb|320x320px|Fluorescent proteins visualize the cell cycle progression. IFP2.0-hGem(1/110) fluorescence is shown in green and highlights the S/G2/M phases. smURFP-hCdtI(30/120) fluorescence is shown in red and highlights the G0/G1 phases.
'''Small ultra red fluorescent protein''' ('''smURFP''') is a class of far-red fluorescent protein developed through [directed evolution](/source/directed_evolution) from a [cyanobacteria](/source/cyanobacteria)l (''[Trichodesmium erythraeum](/source/Trichodesmium_erythraeum)'') [phycobiliprotein](/source/phycobiliprotein), α-[allophycocyanin](/source/allophycocyanin).[{{Cite journal|last1=Rodriguez|first1=Erik A.|last2=Tran|first2=Geraldine N.|last3=Gross|first3=Larry A.|last4=Crisp|first4=Jessica L.|last5=Shu|first5=Xiaokun|last6=Lin|first6=John Y.|last7=Tsien|first7=Roger Y.|date=2016-08-01|title=A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein|journal=Nature Methods|doi=10.1038/nmeth.3935|issn=1548-7105|pmid=27479328|volume=13|issue=9|pages=763–9|pmc=5007177}}][{{Cite patent|country=US|number=20180201655A1|title=Allophycocyanin alpha-subunit evolved labeling proteins (smURFPs).|status=Patent|pubdate=|gdate=2019-12-10|invent1=Rodriguez|invent2=Tran|invent3=Lin|invent4=Ting|inventor1-first=Erik A.|inventor2-first=Geraldine N.|inventor3-first=John Y.|inventor4-first=Richard|url=https://patents.google.com/patent/US20180201655A1/en}}][{{Citation |last1=Mattson |first1=Sara |title=Directed Evolution of Fluorescent Proteins in Bacteria |date=2023 |url=https://link.springer.com/10.1007/978-1-0716-2667-2_4 |work=Fluorescent Proteins |volume=2564 |pages=75–97 |editor-last=Sharma |editor-first=Mayank |place=New York, NY |publisher=Springer US |language=en |doi=10.1007/978-1-0716-2667-2_4 |isbn=978-1-0716-2666-5 |access-date=2022-09-16 |last2=Tran |first2=Geraldine N. |last3=Rodriguez |first3=Erik A.|pmid=36107338 |url-access=subscription }}] Native α-allophycocyanin requires an exogenous protein, known as a [lyase](/source/lyase), to attach the [chromophore](/source/chromophore), phycocyanobilin. Phycocyanobilin is not present in [mammal](/source/mammal)ian [cells](/source/Cell_(biology)). smURFP was evolved to [covalently](/source/covalently) attach phycocyanobilin without a lyase and [fluoresce](/source/Fluorescence), covalently attach [biliverdin](/source/biliverdin) (ubiquitous to mammalian cells) and fluoresce, [blue-shift](/source/Blueshift) fluorescence to match the organic [fluorophore](/source/fluorophore), [Cy5](/source/Cy5), and not inhibit ''[E. coli](/source/Escherichia_coli)'' growth. smURFP was found after 12 rounds of random mutagenesis and manually screening 10,000,000 [bacteria](/source/bacteria)l colonies.
== Properties ==
smURFP is a homodimer with [absorption](/source/Absorption_spectroscopy) and [emission](/source/Emission_spectrum) 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](/source/protein_degradation) [half-life](/source/half-life) of 17 hour and 33 hour without and with chromophore (biliverdin), respectively. This is comparable to the [jellyfish](/source/jellyfish)-derived enhanced green fluorescent protein ([eGFP](/source/Green_fluorescent_protein)) protein degradation half-life of 24 hour.[{{Cite journal|last1=Stack|first1=J. H.|last2=Whitney|first2=M.|last3=Rodems|first3=S. M.|last4=Pollok|first4=B. A.|date=2000-12-01|title=A ubiquitin-based tagging system for controlled modulation of protein stability|journal=Nature Biotechnology|volume=18|issue=12|pages=1298–1302|doi=10.1038/82422|issn=1087-0156|pmid=11101811|s2cid=23741831}}] smURFP is extremely photostable and outperforms [mCherry](/source/mCherry)[{{Cite journal|last1=Shaner|first1=Nathan C.|last2=Campbell|first2=Robert E.|last3=Steinbach|first3=Paul A.|last4=Giepmans|first4=Ben N. G.|last5=Palmer|first5=Amy E.|last6=Tsien|first6=Roger Y.|date=2004-12-01|title=Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein|journal=Nature Biotechnology|volume=22|issue=12|pages=1567–1572|doi=10.1038/nbt1037|issn=1087-0156|pmid=15558047|s2cid=205272166}}] and tdTomato[{{Cite journal |last1=Maiti |first1=Atanu |last2=Buffalo |first2=Cosmo Z. |last3=Saurabh |first3=Saumya |last4=Montecinos-Franjola |first4=Felipe |last5=Hachey |first5=Justin S. |last6=Conlon |first6=William J. |last7=Tran |first7=Geraldine N. |last8=Hassan |first8=Bakar |last9=Walters |first9=Kylie J. |last10=Drobizhev |first10=Mikhail |last11=Moerner |first11=W. E. |last12=Ghosh |first12=Partho |last13=Matsuo |first13=Hiroshi |last14=Tsien |first14=Roger Y. |last15=Lin |first15=John Y. |date=2023-07-12 |title=Structural and photophysical characterization of the small ultra-red fluorescent protein |journal=Nature Communications |language=en |volume=14 |issue=1 |page=4155 |doi=10.1038/s41467-023-39776-9 |pmid=37438348 |issn=2041-1723|pmc=10338489 |bibcode=2023NatCo..14.4155M }}] The [extinction coefficient](/source/Molar_attenuation_coefficient) (180,000 M−1 cm−1) of smURFP is extremely large and has a modest [quantum yield](/source/quantum_yield) (0.18), which makes it comparable biophysical brightness to [eGFP](/source/eGFP) and ~2-fold brighter than most red or far-[red fluorescent proteins](/source/Red_fluorescent_protein) derived from [coral](/source/coral). smURFP has the largest [two-photon](/source/Two-photon_absorption) cross-section measured for a [fluorescent protein](/source/fluorescent_protein). There are two peak cross-sections of 1,060 and 60 [GM](/source/Maria_Goeppert_Mayer) at 820 and 1,196 [nm](/source/Nanometre), respectively.[{{Cite journal |last1=Maiti |first1=Atanu |last2=Buffalo |first2=Cosmo Z. |last3=Saurabh |first3=Saumya |last4=Montecinos-Franjola |first4=Felipe |last5=Hachey |first5=Justin S. |last6=Conlon |first6=William J. |last7=Tran |first7=Geraldine N. |last8=Hassan |first8=Bakar |last9=Walters |first9=Kylie J. |last10=Drobizhev |first10=Mikhail |last11=Moerner |first11=W. E. |last12=Ghosh |first12=Partho |last13=Matsuo |first13=Hiroshi |last14=Tsien |first14=Roger Y. |last15=Lin |first15=John Y. |date=2023-07-12 |title=Structural and photophysical characterization of the small ultra-red fluorescent protein |journal=Nature Communications |language=en |volume=14 |issue=1 |page=4155 |doi=10.1038/s41467-023-39776-9 |pmid=37438348 |issn=2041-1723|pmc=10338489 |bibcode=2023NatCo..14.4155M }}] Despite being a homodimer, all tested N- and C- terminal [fusions](/source/Fusion_protein) show correct cellular localization, including the difficult fusion to [α-tubulin](/source/%CE%B1-tubulin) and [Lamin B1](/source/Lamin_B1) ([Figure](/source/%3AFile%3ASmURFP_Fused_to_Lamin_B1.gif)).[{{Cite journal |last1=Maiti |first1=Atanu |last2=Buffalo |first2=Cosmo Z. |last3=Saurabh |first3=Saumya |last4=Montecinos-Franjola |first4=Felipe |last5=Hachey |first5=Justin S. |last6=Conlon |first6=William J. |last7=Tran |first7=Geraldine N. |last8=Hassan |first8=Bakar |last9=Walters |first9=Kylie J. |last10=Drobizhev |first10=Mikhail |last11=Moerner |first11=W. E. |last12=Ghosh |first12=Partho |last13=Matsuo |first13=Hiroshi |last14=Tsien |first14=Roger Y. |last15=Lin |first15=John Y. |date=2023-07-12 |title=Structural and photophysical characterization of the small ultra-red fluorescent protein |journal=Nature Communications |language=en |volume=14 |issue=1 |page=4155 |doi=10.1038/s41467-023-39776-9 |pmid=37438348 |issn=2041-1723|pmc=10338489 |bibcode=2023NatCo..14.4155M }}] The crystal structure of a smURFP mutant ({{PDB|6FZN}}) was published by Fuenzalida-Werner et al.[{{cite journal |last1=Fuenzalida-Werner |first1=JP |last2=Janowski |first2=R |last3=Mishra |first3=K |last4=Weidenfeld |first4=I |last5=Niessing |first5=D |last6=Ntziachristos |first6=V |last7=Stiel |first7=AC |title=Crystal structure of a biliverdin-bound phycobiliprotein: Interdependence of oligomerization and chromophorylation. |journal=Journal of Structural Biology |date=December 2018 |volume=204 |issue=3 |pages=519–522 |doi=10.1016/j.jsb.2018.09.013 |pmid=30287387|s2cid=52919137 }}] The mutants show significantly larger chromophore pockets and protein volume, which results in diminished quantum yield.[{{Cite journal |last1=Maiti |first1=Atanu |last2=Buffalo |first2=Cosmo Z. |last3=Saurabh |first3=Saumya |last4=Montecinos-Franjola |first4=Felipe |last5=Hachey |first5=Justin S. |last6=Conlon |first6=William J. |last7=Tran |first7=Geraldine N. |last8=Hassan |first8=Bakar |last9=Walters |first9=Kylie J. |last10=Drobizhev |first10=Mikhail |last11=Moerner |first11=W. E. |last12=Ghosh |first12=Partho |last13=Matsuo |first13=Hiroshi |last14=Tsien |first14=Roger Y. |last15=Lin |first15=John Y. |date=2023-07-12 |title=Structural and photophysical characterization of the small ultra-red fluorescent protein |journal=Nature Communications |language=en |volume=14 |issue=1 |page=4155 |doi=10.1038/s41467-023-39776-9 |pmid=37438348 |issn=2041-1723|pmc=10338489 |bibcode=2023NatCo..14.4155M }}] A 2020 review discusses recent applications of smURFP as a genetically encoded or exogenous probe for ''in vivo'' imaging and discusses problems with [biliverdin](/source/biliverdin) availability.[{{Cite journal|last1=Montecinos-Franjola|first1=Felipe|last2=Lin|first2=John Y.|last3=Rodriguez|first3=Erik A.|date=2020-11-16|title=Fluorescent proteins for in vivo imaging, where's the biliverdin?|url=https://portlandpress.com/biochemsoctrans/article/doi/10.1042/BST20200444/226972/Fluorescent-proteins-for-in-vivo-imaging-wheres|journal=Biochemical Society Transactions|volume=48 |issue=6 |language=en|pages=2657–2667|doi=10.1042/BST20200444|pmid=33196077 |s2cid=226971864 |issn=0300-5127|url-access=subscription}}]
thumb|321x321px|Image shows ''E. coli'' expressing smURFP, pelleting of ''E. coli'', removal of media, ''E. coli'' lysis, smURFP binding to NiNTA, smURFP elution, and buffer exchange.
== smURFP used as nanoparticles, exogenous probes, and ''in vitro'' assays ==
Free smURFP is 2-3 nm in diameter. smURFP [nanoparticle](/source/nanoparticle)s of ~10-14 nm diameter can be synthesized in an oil and water [emulsion](/source/emulsion) and remain fluorescent. These fluorescent [protein nanoparticles](/source/protein_nanoparticles) are stable in living mice and useful for [non-invasive](/source/Non-invasive_procedure) [tumor](/source/Neoplasm) [fluorescence imaging](/source/fluorescence_imaging).[{{Cite journal|last1=An|first1=Feifei|last2=Chen|first2=Nandi|last3=Conlon|first3=William J.|last4=Hachey|first4=Justin S.|last5=Xin|first5=Jingqi|last6=Aras|first6=Omer|last7=Rodriguez|first7=Erik A.|last8=Ting|first8=Richard|date=February 2020|title=Small ultra-red fluorescent protein nanoparticles as exogenous probes for noninvasive tumor imaging in vivo|journal=International Journal of Biological Macromolecules|volume=153|language=en|pages=100–106|doi=10.1016/j.ijbiomac.2020.02.253|pmid=32105698|pmc=7493049}}]
Purified smURFP survives [ultrasound](/source/ultrasound) and [fixation](/source/Fixation_(histology)) to allow fluorescence imaging of [macromolecule](/source/macromolecule) delivery by ultrasound into [corneas](/source/Cornea).[{{Cite journal |last1=Almogbil |first1=Hanaa H. |last2=Montecinos-Franjola |first2=Felipe |last3=Daszynski |first3=Camille |last4=Conlon |first4=William J. |last5=Hachey |first5=Justin S. |last6=Corazza |first6=Giavanna |last7=Rodriguez |first7=Erik A. |last8=Zderic |first8=Vesna |date=2022-08-01 |title=Therapeutic Ultrasound for Topical Corneal Delivery of Macromolecules |journal=Translational Vision Science & Technology |language=en |volume=11 |issue=8 |page=23 |doi=10.1167/tvst.11.8.23 |pmid=35998058 |pmc=9424970 |s2cid=251743679 |issn=2164-2591|doi-access=free }}]
Free smURFP, purified protein and not genetically encoded, can be encapsulated into [virus](/source/virus)es and used for non-invasive, fluorescence imaging of [biodistribution](/source/biodistribution) in living mice.[{{cite journal |last1=Herbert |first1=Fabian C. |last2=Brohlin |first2=Olivia R. |last3=Galbraith |first3=Tyler |last4=Benjamin |first4=Candace |last5=Reyes |first5=Cesar A. |last6=Luzuriaga |first6=Michael A. |last7=Shahrivarkevishahi |first7=Arezoo |last8=Gassensmith |first8=Jeremiah J. |title=Supramolecular Encapsulation of Small-Ultrared Fluorescent Proteins in Virus-Like Nanoparticles for Noninvasive In Vivo Imaging Agents |journal=Bioconjugate Chemistry |date=20 May 2020 |volume=31 |issue=5 |pages=1529–1536 |doi=10.1021/acs.bioconjchem.0c00190|pmid=32343135 }}][{{Cite journal|last1=Herbert|first1=Fabian C.|last2=Brohlin|first2=Olivia R.|last3=Galbraith|first3=Tyler|last4=Benjamin|first4=Candace|last5=Reyes|first5=Cesar A.|last6=Luzuriaga|first6=Michael A.|last7=Shahrivarkevishahi|first7=Arezoo|last8=Gassensmith|first8=Jeremiah J.|date=2020-05-07|title=Supramolecular Encapsulation of Small-Ultrared Fluorescent Proteins in Virus-Like Nanoparticles for Noninvasive In Vivo Imaging Agents|journal=Bioconjugate Chemistry|volume=31|issue=5|language=en|pages=1529–1536|doi=10.1021/acs.bioconjchem.0c00190|pmid=32343135|issn=1043-1802|url=https://figshare.com/articles/Supramolecular_Encapsulation_of_Small-Ultra_Red_Fluorescent_Proteins_in_Virus-Like_Nanoparticles_for_Non-Invasive_In_Vivo_Imaging_Agents/12067851}}][{{Cite journal |last1=Trashi |first1=Ikeda |last2=Durbacz |first2=Mateusz Z. |last3=Trashi |first3=Orikeda |last4=Wijesundara |first4=Yalini H. |last5=Ehrman |first5=Ryanne N. |last6=Chiev |first6=Alyssa C. |last7=Darwin |first7=Cary B. |last8=Herbert |first8=Fabian C. |last9=Gadhvi |first9=Jashkaran |last10=Nisco |first10=Nicole J. De |last11=Nielsen |first11=Steven O. |last12=Gassensmith |first12=Jeremiah J. |date=2023-04-25 |title=Self-assembly of a fluorescent virus-like particle for imaging in tissues with high autofluorescence |url=https://pubs.rsc.org/en/content/articlelanding/2023/tb/d3tb00469d |journal=Journal of Materials Chemistry B |volume=11 |issue=20 |pages=4445–4452 |language=en |doi=10.1039/D3TB00469D |pmid=37144595 |issn=2050-7518|url-access=subscription }}]
smURFP covalently attaches [biliverdin](/source/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](/source/Serum_(blood)).[{{Cite journal|last1=Zhu|first1=Xiaqing|last2=Feng|first2=Shuren|last3=Jiang|first3=Zhongyi|last4=Zhang|first4=Huayue|last5=Wang|first5=Yanyan|last6=Yang|first6=Haitao|last7=Wang|first7=Zefang|date=August 2021|title=An ultra-red fluorescent biosensor for highly sensitive and rapid detection of biliverdin|url=https://linkinghub.elsevier.com/retrieve/pii/S0003267021005353|journal=Analytica Chimica Acta|language=en|volume=1174|article-number=338709|doi=10.1016/j.aca.2021.338709|pmid=34247733 |bibcode=2021AcAC.117438709Z |s2cid=235796133 |url-access=subscription}}] smURFP allows for the creation of ''[in vitro](/source/in_vitro)'' assays to detect [enzyme](/source/enzyme) activity. An [assay](/source/assay) was developed for [thrombin](/source/thrombin) with a detection range of 1.07 aM–0.01 mM and a limit of detection of 0.2 aM.[{{Cite journal|last1=Zhang|first1=Huayue|last2=Yang|first2=Lu|last3=Zhu|first3=Xiaqing|last4=Wang|first4=Yanyan|last5=Yang|first5=Haitao|last6=Wang|first6=Zefang|date=2020-05-13|title=A Rapid and Ultrasensitive Thrombin Biosensor Based on a Rationally Designed Trifunctional Protein|journal=Advanced Healthcare Materials|volume=9|issue=12|language=en|article-number=2000364|doi=10.1002/adhm.202000364|pmid=32406199|s2cid=218633091 |issn=2192-2640}}]
Tandem dimer smURFP (TDsmURFP) was used as an exogenous [fluorescent marker](/source/Fluorescent_tag) to label the seven-[transmembrane receptor](/source/Cell_surface_receptor) [Smoothened](/source/Smoothened) (SMO). TDsmURFP was purified from ''[E. coli](/source/Escherichia_coli)'' and attached to SMO by [sortase](/source/sortase)-mediated conjugation for [fluorescence-activated cell sorting (FACS)](/source/Fluorescence-activated_cell_sorting).[{{Cite journal|last1=Deshpande|first1=Ishan|last2=Liang|first2=Jiahao|last3=Hedeen|first3=Danielle|last4=Roberts|first4=Kelsey J.|last5=Zhang|first5=Yunxiao|last6=Ha|first6=Betty|last7=Latorraca|first7=Naomi R.|last8=Faust|first8=Bryan|last9=Dror|first9=Ron O.|last10=Beachy|first10=Philip A.|last11=Myers|first11=Benjamin R.|date=July 2019|title=Smoothened stimulation by membrane sterols drives Hedgehog pathway activity|journal=Nature|language=en|volume=571|issue=7764|pages=284–288|doi=10.1038/s41586-019-1355-4|issn=1476-4687|pmc=6709672|pmid=31263273}}] 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 ==
thumb|326x326px|The small Ultra-Red Fluorescent Protein (smURFP) is a self-labeling protein. The substrate is fluorogenic, fluoresces when attached, and quenches fluorescent cargo. The smURFP-tag[{{Cite journal|last1=Machado|first1=John-Hanson|last2=Ting|first2=Richard|last3=Lin|first3=John Y.|last4=Rodriguez|first4=Erik A.|date=2021|title=A self-labeling protein based on the small ultra-red fluorescent protein, smURFP|journal=RSC Chemical Biology|volume=2 |issue=4 |language=en|pages=1221–1226|pmid=34458834 | doi=10.1039/D1CB00127B | pmc=8341759|issn=2633-0679|doi-access=free}}] has novel properties for tool development.
The small Ultra-Red Fluorescent Protein (smURFP) is a self-labeling protein like [Halo-](/source/HaloTag), [SNAP](/source/SNAP-tag)-, and [CLIP](/source/CLIP-tag)-tags. The smURFP-tag accepts a [biliverdin](/source/biliverdin) substrate modified on a [carboxylate](/source/carboxylate) with a [polyethylene glycol](/source/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](/source/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](/source/Quenching_(fluorescence)) [fluorescein](/source/fluorescein) cargo to allow for imaging without substrate removal. Biliverdin modification on a single carboxylate creates a neutral molecule that passes the [outer](/source/Cell_membrane) and [nuclear membrane](/source/Nuclear_envelope) of mammalian [cells](/source/Cell_(biology)).[{{Cite journal|last1=Machado|first1=John-Hanson|last2=Ting|first2=Richard|last3=Lin|first3=John Y.|last4=Rodriguez|first4=Erik A.|date=2021|title=A self-labeling protein based on the small ultra-red fluorescent protein, smURFP.|journal=RSC Chemical Biology|volume=2 |issue=4 |language=en|pages=1221–1226|doi=10.1039/D1CB00127B|pmid=34458834 |pmc=8341759 |issn=2633-0679|doi-access=free}}]
== Chromophore availability in cells and mice ==
thumb|321x321px|smURFP was genetically fused to human, lamin B1 to show the nuclear envelope with fluorescence. Localization of the Lamin B1 protein changes during different phases of the cell cycle.
Despite showing comparable biophysical brightness to [eGFP](/source/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](/source/biliverdin) was not comparable to eGFP. Biliverdin has two [carboxylate](/source/carboxylate)s at neutral [pH](/source/pH) and this is inhibiting cellular entry. Biliverdin dimethyl ester is a more [hydrophobic](/source/Hydrophobic_effect) analog and readily crosses the cellular [membrane](/source/membrane). smURFP with biliverdin dimethyl ester shows comparable fluorescence to [eGFP](/source/eGFP) in [cells](/source/Cell_(biology)) and is brighter than [bacteria](/source/bacteria)l [phytochrome](/source/phytochrome) fluorescent proteins.
The free [chromophore](/source/chromophore) can be differentiated from chromophore attached to smURFP by fluorescence lifetime imaging ([FLIM](/source/Fluorescence-lifetime_imaging_microscopy)) 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](/source/Nanosecond).[{{Cite journal |last1=Maiti |first1=Atanu |last2=Buffalo |first2=Cosmo Z. |last3=Saurabh |first3=Saumya |last4=Montecinos-Franjola |first4=Felipe |last5=Hachey |first5=Justin S. |last6=Conlon |first6=William J. |last7=Tran |first7=Geraldine N. |last8=Hassan |first8=Bakar |last9=Walters |first9=Kylie J. |last10=Drobizhev |first10=Mikhail |last11=Moerner |first11=W. E. |last12=Ghosh |first12=Partho |last13=Matsuo |first13=Hiroshi |last14=Tsien |first14=Roger Y. |last15=Lin |first15=John Y. |date=2023-07-12 |title=Structural and photophysical characterization of the small ultra-red fluorescent protein |journal=Nature Communications |language=en |volume=14 |issue=1 |page=4155 |doi=10.1038/s41467-023-39776-9 |pmid=37438348 |issn=2041-1723|pmc=10338489 |bibcode=2023NatCo..14.4155M }}]
[[File:SmURFP Expressed in Neuronal Culture.tif|thumb|316x316px|smURFP expressed in neuronal culture does not show aggregation in lysosomes, which was seen with the fluorescent protein, [mCherry](/source/mCherry).]]
In mice, smURFP fluorescence is visible in [HT1080](/source/HT1080) tumor [xenografts](/source/xenografts) without exogenous biliverdin, but fluorescence is less than coral-derived red fluorescent proteins, [mCherry](/source/mCherry)[{{Cite journal|last1=Chu|first1=Jun|last2=Haynes|first2=Russell D|last3=Corbel|first3=Stéphane Y|last4=Li|first4=Pengpeng|last5=González-González|first5=Emilio|last6=Burg|first6=John S|last7=Ataie|first7=Niloufar J|last8=Lam|first8=Amy J|last9=Cranfill|first9=Paula J|title=Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein|journal=Nature Methods|volume=11|issue=5|pages=572–578|doi=10.1038/nmeth.2888|pmc=4008650|pmid=24633408|year=2014}}] Visible fluorescence is not always usable fluorescence and fluorescent proteins should always be compared to other useful, genetically encoded fluorescent proteins. [Intravenous injection](/source/Intravenous_injection) of exogenous [biliverdin](/source/biliverdin) or biliverdin dimethyl ester does not increase fluorescence of smURFP expressed in [tumors](/source/tumors) after 1 to 24 hours. [Mass spectrometry](/source/Mass_spectrometry) showed that the [ester groups](/source/Ester) were rapidly removed from biliverdin dimethyl ester. Addition of 25 μM [biliverdin](/source/biliverdin) or biliverdin dimethyl ester dramatically increased fluorescence of [excised](/source/Surgery) [tumors](/source/tumors) and smURFP is present without chromophore. Further research is necessary to optimize [chromophore](/source/chromophore) availability in mice to obtain fluorescence comparable or greater than [coral](/source/coral)-derived red fluorescent proteins.
== Adding chromophore to cells ==
Biliverdin dimethyl ester, [biliverdin](/source/biliverdin), or [phycocyanobilin](/source/phycocyanobilin) is dissolved in [DMSO](/source/Dimethyl_sulfoxide) at a [concentration](/source/concentration) of 5 mM. The solution is very dark and pipette vigorously to ensure all is dissolved. Biliverdin dimethyl ester is not soluble in common [buffers](/source/Buffer_solution), including [phosphate buffered saline](/source/Phosphate-buffered_saline) (PBS) or [Hank's balanced salt solution](/source/Hanks'_salts) (HBSS). Add 1-5 μM biliverdin dimethyl ester in media or buffer containing 10% [fetal bovine serum](/source/fetal_bovine_serum) (FBS). Add 25 μM [biliverdin](/source/biliverdin) (not as membrane permeant) to [cells](/source/Cell_(biology)). [Biliverdin](/source/Biliverdin) does not saturate the smURFP sites and does not achieve maximum fluorescence intensity. Biliverdin dimethyl ester should be used to get maximum fluorescence intensity. Incubate smURFP with [chromophore](/source/chromophore) for as long as possible to increase protein accumulation caused by enhanced [protein stability](/source/protein_stability) with [chromophore](/source/chromophore). Leave [chromophore](/source/chromophore) for a minimum of 3 hours and 24 h is recommended. Remove [chromophore](/source/chromophore), wash with media containing 10% [FBS](/source/Fetal_bovine_serum), and image in media lacking [phenol red](/source/phenol_red) or imaging [buffer](/source/Buffer_solution).
== smURFP genetically encoded biosensors ==
thumb|318x318px|Far Red & Near Infrared FUCCI visualizes the birth of a multinucleated cell, which is common in many cancer cells.''[Kinase](/source/Kinase) [FRET](/source/F%C3%B6rster_resonance_energy_transfer) sensor.'' smURFP is a useful acceptor for many red [fluorescent proteins](/source/Green_fluorescent_protein) due to spectral overlap. A rationally designed red fluorescent protein, stagRFP, allows for easier creation of [FRET](/source/F%C3%B6rster_resonance_energy_transfer) sensors. stagRFP is a useful FRET donor to the far-red acceptor smURFP and an [ERK](/source/Extracellular_signal-regulated_kinases) [kinase](/source/kinase) [FRET](/source/F%C3%B6rster_resonance_energy_transfer) reporter was created with an average response of ~15%. The new sensor allowed for simultaneous visualization of three kinases, [Src](/source/Proto-oncogene_tyrosine-protein_kinase_Src), [Akt](/source/Protein_kinase_B), [ERK](/source/Extracellular_signal-regulated_kinases), in a single [cell](/source/Cell_(biology)).[{{Cite journal|last1=Mo|first1=Gary C. H.|last2=Posner|first2=Clara|last3=Rodriguez|first3=Erik A.|last4=Sun|first4=Tengqian|last5=Zhang|first5=Jin|date=December 2020|title=A rationally enhanced red fluorescent protein expands the utility of FRET biosensors|journal=Nature Communications|language=en|volume=11|issue=1|page=1848|doi=10.1038/s41467-020-15687-x|pmid=32296061|pmc=7160135|bibcode=2020NatCo..11.1848M|issn=2041-1723|doi-access=free}}]
''Fluorescently imaging the [cell cycle](/source/cell_cycle).'' Pioneering work by Atsushi Miyawaki and coworkers developed the fluorescent ubiquitination-based cell cycle indicator (FUCCI), which enables [fluorescence](/source/fluorescence) imaging of the cell cycle. Originally, a [green fluorescent protein](/source/green_fluorescent_protein), mAG, was fused to hGem(1/110) and an orange [fluorescent protein](/source/fluorescent_protein) (mKO2) was fused to hCdt1(30/120). Note, these fusions are fragments that contain a [nuclear localization signal](/source/Nuclear_Localization_Signal) and [ubiquitination](/source/ubiquitination) sites for [degradation](/source/Protein_degradation), but are not functional proteins. The [green fluorescent protein](/source/green_fluorescent_protein) is made during the S, G2, or M phase and degraded during the G0 or G1 phase, while the orange fluorescent protein is made during the G0 or G1 phase and destroyed during the S, G2, or M phase.[{{Cite journal|last1=Sakaue-Sawano|first1=Asako|last2=Kurokawa|first2=Hiroshi|last3=Morimura|first3=Toshifumi|last4=Hanyu|first4=Aki|last5=Hama|first5=Hiroshi|last6=Osawa|first6=Hatsuki|last7=Kashiwagi|first7=Saori|last8=Fukami|first8=Kiyoko|last9=Miyata|first9=Takaki|date=2008-02-08|title=Visualizing spatiotemporal dynamics of multicellular cell-cycle progression|journal=Cell|volume=132|issue=3|pages=487–498|doi=10.1016/j.cell.2007.12.033|issn=1097-4172|pmid=18267078|s2cid=15704902|doi-access=free}}] A far-red and near-infrared FUCCI was developed using a [cyanobacteria](/source/cyanobacteria)-derived fluorescent protein (smURFP) and a [bacteriophytochrome](/source/Phytochrome)-derived [fluorescent protein](/source/fluorescent_protein) ([http://www.nature.com/nmeth/journal/vaop/ncurrent/fig_tab/nmeth.3935_SV2.html movie found at this link]).