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 evolved from a
cyanobacterial (
Trichodesmium erythraeum )
phycobiliprotein , α-
allophycocyanin .
[1]
[2]
[3] 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.
[4] smURFP is extremely photostable and out performs
mCherry
[5] and tdTomato
[5] in living cells.
Single-molecule smURFPs emit twice as many
photons before
photobleaching than small-molecule dyes AlexaFluor647 and Cyanine5.
[6] 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.
[7] 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 ).
[1] smURFP is named after
the Smurfs , due to its light blue appearance in white light.
[1]
An Excel sheet of the smURFP absorbance, excitation, and emission spectra can be downloaded
here . 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 .
[8] The
crystal structure of a smURFP mutant (
PDB :
6FZN ) was published in
Fuenzalida-Werner et al .
[9] The mutants show significantly larger chromophore pockets and protein volume, which results in diminished quantum yield.
[10] 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.
[11]
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
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.
[12]
Purified smURFP survives
ultrasound and
fixation to allow
fluorescence imaging of
macromolecule delivery by
ultrasound into
corneas .
[13]
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.
[14]
[15]
[16]
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 .
[17] 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.
[18]
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) .
[19] 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. The substrate is fluorogenic, fluoresces when attached, and quenches fluorescent cargo. The smURFP-tag
[20] has novel properties for tool development.
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 .
[21]
Chromophore availability in cells and mice
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 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 .
[22]
smURFP expressed in neuronal culture does not show aggregation in lysosomes, which was seen with the fluorescent protein,
mCherry .
In mice, smURFP
fluorescence is visible in
HT1080
tumor
xenografts without exogenous
biliverdin , but
fluorescence is less than
coral -derived red
fluorescent proteins ,
mCherry
[5] and mCardinal.
[23] Visible
fluorescence is not always usable
fluorescence and
fluorescent proteins should always be compared to other useful,
genetically encoded
fluorescent proteins .
Intravenous injection of exogenous
biliverdin or
biliverdin dimethyl ester does not increase
fluorescence of smURFP expressed in
tumors after 1 to 24 hours.
Mass spectrometry showed that the
ester groups were rapidly removed from
biliverdin dimethyl ester . Addition of 25 μM
biliverdin or
biliverdin dimethyl ester dramatically increased
fluorescence of
excised
tumors and smURFP is present without
chromophore . Further research is necessary to optimize
chromophore availability in mice to obtain
fluorescence comparable or greater than
coral -derived red
fluorescent proteins .
Adding chromophore to cells
Biliverdin dimethyl ester ,
biliverdin , and
phycocyanobilin are commercially available from
Frontier Scientific .
Biliverdin dimethyl ester ,
biliverdin , or
phycocyanobilin is dissolved in
DMSO at a
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 , including
phosphate buffered saline (PBS) or
Hank's balanced salt solution (HBSS). Add 1-5 μM
biliverdin dimethyl ester in media or buffer containing 10%
fetal bovine serum (FBS). Add 25 μM
biliverdin (not as membrane permeant) to
cells .
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 for as long as possible to increase protein accumulation caused by enhanced
protein stability with
chromophore . Leave
chromophore for a minimum of 3 hours and 24 h is recommended. Remove
chromophore , wash with media containing 10%
FBS , and image in media lacking
phenol red or imaging
buffer .
smURFP genetically encoded biosensors
Far Red & Near Infrared FUCCI visualizes the birth of a multinucleated cell, which is common in many cancer cells.
Kinase
FRET sensor. smURFP is a useful acceptor for many red
fluorescent proteins due to spectral overlap. A rationally designed red fluorescent protein, stagRFP, allows for easier creation of
FRET sensors. stagRFP is a useful FRET donor to the far-red acceptor smURFP and an
ERK
kinase
FRET reporter was created with an average response of ~15%. The new sensor allowed for simultaneous visualization of three kinases,
Src ,
Akt ,
ERK , in a single
cell .
[24]
Fluorescently imaging the
cell cycle . Pioneering work by Atsushi Miyawaki and coworkers developed the fluorescent ubiquitination-based cell cycle indicator (
FUCCI ), which enables
fluorescence imaging of the cell cycle. Originally, a
green fluorescent protein , mAG, was fused to hGem(1/110) and an orange
fluorescent protein (mKO2 ) was fused to hCdt1(30/120). Note, these fusions are fragments that contain a
nuclear localization signal and
ubiquitination sites for
degradation , but are not functional proteins. The
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.
[25] A far-red and near-infrared FUCCI was developed using a
cyanobacteria -derived
fluorescent protein (smURFP) and a
bacteriophytochrome -derived
fluorescent protein (
movie found at this link ).
[1]
smURFP (light-blue) expressed in E. coli .
References
^
a
b
c
d Rodriguez, Erik A.; Tran, Geraldine N.; Gross, Larry A.; Crisp, Jessica L.; Shu, Xiaokun; Lin, John Y.; Tsien, Roger Y. (2016-08-01).
"A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein" . Nature Methods . 13 (9): 763–9.
doi :
10.1038/nmeth.3935 .
ISSN
1548-7105 .
PMC
5007177 .
PMID
27479328 .
^
US Patent 20180201655A1 , Rodriguez, Erik A.; Tran, Geraldine N. & Lin, John Y. et al., "Allophycocyanin alpha-subunit evolved labeling proteins (smURFPs).", issued 2019-12-10
^ Mattson, Sara; Tran, Geraldine N.; Rodriguez, Erik A. (2023), Sharma, Mayank (ed.),
"Directed Evolution of Fluorescent Proteins in Bacteria" , Fluorescent Proteins , vol. 2564, New York, NY: Springer US, pp. 75–97,
doi :
10.1007/978-1-0716-2667-2_4 ,
ISBN
978-1-0716-2666-5 ,
PMID
36107338 , retrieved 2022-09-16
^ Stack, J. H.; Whitney, M.; Rodems, S. M.; Pollok, B. A. (2000-12-01). "A ubiquitin-based tagging system for controlled modulation of protein stability". Nature Biotechnology . 18 (12): 1298–1302.
doi :
10.1038/82422 .
ISSN
1087-0156 .
PMID
11101811 .
S2CID
23741831 .
^
a
b
c Shaner, Nathan C.; Campbell, Robert E.; Steinbach, Paul A.; Giepmans, Ben N. G.; Palmer, Amy E.; Tsien, Roger Y. (2004-12-01). "Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein". Nature Biotechnology . 22 (12): 1567–1572.
doi :
10.1038/nbt1037 .
ISSN
1087-0156 .
PMID
15558047 .
S2CID
205272166 .
^ Maiti, Atanu; Buffalo, Cosmo Z.; Saurabh, Saumya; Montecinos-Franjola, Felipe; Hachey, Justin S.; Conlon, William J.; Tran, Geraldine N.; Hassan, Bakar; Walters, Kylie J.; Drobizhev, Mikhail; Moerner, W. E.; Ghosh, Partho; Matsuo, Hiroshi; Tsien, Roger Y.; Lin, John Y. (2023-07-12).
"Structural and photophysical characterization of the small ultra-red fluorescent protein" . Nature Communications . 14 (1): 4155.
doi :
10.1038/s41467-023-39776-9 .
ISSN
2041-1723 .
PMC
10338489 .
^ Maiti, Atanu; Buffalo, Cosmo Z.; Saurabh, Saumya; Montecinos-Franjola, Felipe; Hachey, Justin S.; Conlon, William J.; Tran, Geraldine N.; Hassan, Bakar; Walters, Kylie J.; Drobizhev, Mikhail; Moerner, W. E.; Ghosh, Partho; Matsuo, Hiroshi; Tsien, Roger Y.; Lin, John Y. (2023-07-12).
"Structural and photophysical characterization of the small ultra-red fluorescent protein" . Nature Communications . 14 (1): 4155.
doi :
10.1038/s41467-023-39776-9 .
ISSN
2041-1723 .
PMC
10338489 .
^ Maiti, Atanu; Buffalo, Cosmo Z.; Saurabh, Saumya; Montecinos-Franjola, Felipe; Hachey, Justin S.; Conlon, William J.; Tran, Geraldine N.; Hassan, Bakar; Walters, Kylie J.; Drobizhev, Mikhail; Moerner, W. E.; Ghosh, Partho; Matsuo, Hiroshi; Tsien, Roger Y.; Lin, John Y. (2023-07-12).
"Structural and photophysical characterization of the small ultra-red fluorescent protein" . Nature Communications . 14 (1): 4155.
doi :
10.1038/s41467-023-39776-9 .
ISSN
2041-1723 .
PMC
10338489 .
^ Fuenzalida-Werner, JP; Janowski, R; Mishra, K; Weidenfeld, I; Niessing, D; Ntziachristos, V; Stiel, AC (December 2018). "Crystal structure of a biliverdin-bound phycobiliprotein: Interdependence of oligomerization and chromophorylation". Journal of Structural Biology . 204 (3): 519–522.
doi :
10.1016/j.jsb.2018.09.013 .
PMID
30287387 .
S2CID
52919137 .
^ Maiti, Atanu; Buffalo, Cosmo Z.; Saurabh, Saumya; Montecinos-Franjola, Felipe; Hachey, Justin S.; Conlon, William J.; Tran, Geraldine N.; Hassan, Bakar; Walters, Kylie J.; Drobizhev, Mikhail; Moerner, W. E.; Ghosh, Partho; Matsuo, Hiroshi; Tsien, Roger Y.; Lin, John Y. (2023-07-12).
"Structural and photophysical characterization of the small ultra-red fluorescent protein" . Nature Communications . 14 (1): 4155.
doi :
10.1038/s41467-023-39776-9 .
ISSN
2041-1723 .
PMC
10338489 .
^ Montecinos-Franjola, Felipe; Lin, John Y.; Rodriguez, Erik A. (2020-11-16).
"Fluorescent proteins for in vivo imaging, where's the biliverdin?" . Biochemical Society Transactions . 48 (6): 2657–2667.
doi :
10.1042/BST20200444 .
ISSN
0300-5127 .
PMID
33196077 .
S2CID
226971864 .
^ An, Feifei; Chen, Nandi; Conlon, William J.; Hachey, Justin S.; Xin, Jingqi; Aras, Omer; Rodriguez, Erik A.; Ting, Richard (February 2020).
"Small ultra-red fluorescent protein nanoparticles as exogenous probes for noninvasive tumor imaging in vivo" . International Journal of Biological Macromolecules . 153 : 100–106.
doi :
10.1016/j.ijbiomac.2020.02.253 .
PMC
7493049 .
PMID
32105698 .
^ Almogbil, Hanaa H.; Montecinos-Franjola, Felipe; Daszynski, Camille; Conlon, William J.; Hachey, Justin S.; Corazza, Giavanna; Rodriguez, Erik A.; Zderic, Vesna (2022-08-01).
"Therapeutic Ultrasound for Topical Corneal Delivery of Macromolecules" . Translational Vision Science & Technology . 11 (8): 23.
doi :
10.1167/tvst.11.8.23 .
ISSN
2164-2591 .
PMC
9424970 .
PMID
35998058 .
S2CID
251743679 .
^ Herbert, Fabian C.; Brohlin, Olivia; Galbraith, Tyler; Benjamin, Candace; Reyes, Cesar A.; Luzuriaga, Michael A.; Shahrivarkevishahi, Arezoo; Gassensmith, Jeremiah J. (2020-04-03).
"Supramolecular Encapsulation of Small-Ultra Red Fluorescent Proteins in Virus-Like Nanoparticles for Non-Invasive In Vivo Imaging Agents" .
doi :
10.26434/chemrxiv.12067851.v1 .
S2CID
216595590 .
^ Herbert, Fabian C.; Brohlin, Olivia R.; Galbraith, Tyler; Benjamin, Candace; Reyes, Cesar A.; Luzuriaga, Michael A.; Shahrivarkevishahi, Arezoo; Gassensmith, Jeremiah J. (2020-05-07).
"Supramolecular Encapsulation of Small-Ultrared Fluorescent Proteins in Virus-Like Nanoparticles for Noninvasive In Vivo Imaging Agents" . Bioconjugate Chemistry . 31 (5): 1529–1536.
doi :
10.1021/acs.bioconjchem.0c00190 .
ISSN
1043-1802 .
PMID
32343135 .
^ Trashi, Ikeda; Durbacz, Mateusz Z.; Trashi, Orikeda; Wijesundara, Yalini H.; Ehrman, Ryanne N.; Chiev, Alyssa C.; Darwin, Cary B.; Herbert, Fabian C.; Gadhvi, Jashkaran; Nisco, Nicole J. De; Nielsen, Steven O.; Gassensmith, Jeremiah J. (2023-04-25).
"Self-assembly of a fluorescent virus-like particle for imaging in tissues with high autofluorescence" . Journal of Materials Chemistry B .
doi :
10.1039/D3TB00469D .
ISSN
2050-7518 .
^ Zhu, Xiaqing; Feng, Shuren; Jiang, Zhongyi; Zhang, Huayue; Wang, Yanyan; Yang, Haitao; Wang, Zefang (August 2021).
"An ultra-red fluorescent biosensor for highly sensitive and rapid detection of biliverdin" . Analytica Chimica Acta . 1174 : 338709.
doi :
10.1016/j.aca.2021.338709 .
PMID
34247733 .
S2CID
235796133 .
^ Zhang, Huayue; Yang, Lu; Zhu, Xiaqing; Wang, Yanyan; Yang, Haitao; Wang, Zefang (2020-05-13). "A Rapid and Ultrasensitive Thrombin Biosensor Based on a Rationally Designed Trifunctional Protein". Advanced Healthcare Materials . 9 (12): 2000364.
doi :
10.1002/adhm.202000364 .
ISSN
2192-2640 .
PMID
32406199 .
S2CID
218633091 .
^ Deshpande, Ishan; Liang, Jiahao; Hedeen, Danielle; Roberts, Kelsey J.; Zhang, Yunxiao; Ha, Betty; Latorraca, Naomi R.; Faust, Bryan; Dror, Ron O.; Beachy, Philip A.; Myers, Benjamin R. (July 2019).
"Smoothened stimulation by membrane sterols drives Hedgehog pathway activity" . Nature . 571 (7764): 284–288.
doi :
10.1038/s41586-019-1355-4 .
ISSN
1476-4687 .
PMC
6709672 .
PMID
31263273 .
^ Machado, John-Hanson; Ting, Richard; Lin, John Y.; Rodriguez, Erik A. (2021).
"A self-labeling protein based on the small ultra-red fluorescent protein, smURFP" . RSC Chemical Biology . 2 (4): 1221–1226.
doi :
10.1039/D1CB00127B .
ISSN
2633-0679 .
PMC
8341759 .
PMID
34458834 .
^ Machado, John-Hanson; Ting, Richard; Lin, John Y.; Rodriguez, Erik A. (2021).
"A self-labeling protein based on the small ultra-red fluorescent protein, smURFP" . RSC Chemical Biology . 2 (4): 1221–1226.
doi :
10.1039/D1CB00127B .
ISSN
2633-0679 .
PMC
8341759 .
PMID
34458834 .
^ Maiti, Atanu; Buffalo, Cosmo Z.; Saurabh, Saumya; Montecinos-Franjola, Felipe; Hachey, Justin S.; Conlon, William J.; Tran, Geraldine N.; Hassan, Bakar; Walters, Kylie J.; Drobizhev, Mikhail; Moerner, W. E.; Ghosh, Partho; Matsuo, Hiroshi; Tsien, Roger Y.; Lin, John Y. (2023-07-12).
"Structural and photophysical characterization of the small ultra-red fluorescent protein" . Nature Communications . 14 (1): 4155.
doi :
10.1038/s41467-023-39776-9 .
ISSN
2041-1723 .
PMC
10338489 .
^ Chu, Jun; Haynes, Russell D; Corbel, Stéphane Y; Li, Pengpeng; González-González, Emilio; Burg, John S; Ataie, Niloufar J; Lam, Amy J; Cranfill, Paula J (2014).
"Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein" . Nature Methods . 11 (5): 572–578.
doi :
10.1038/nmeth.2888 .
PMC
4008650 .
PMID
24633408 .
^ Mo, Gary C. H.; Posner, Clara; Rodriguez, Erik A.; Sun, Tengqian; Zhang, Jin (December 2020).
"A rationally enhanced red fluorescent protein expands the utility of FRET biosensors" . Nature Communications . 11 (1): 1848.
Bibcode :
2020NatCo..11.1848M .
doi :
10.1038/s41467-020-15687-x .
ISSN
2041-1723 .
PMC
7160135 .
PMID
32296061 .
^ Sakaue-Sawano, Asako; Kurokawa, Hiroshi; Morimura, Toshifumi; Hanyu, Aki; Hama, Hiroshi; Osawa, Hatsuki; Kashiwagi, Saori; Fukami, Kiyoko; Miyata, Takaki (2008-02-08).
"Visualizing spatiotemporal dynamics of multicellular cell-cycle progression" . Cell . 132 (3): 487–498.
doi :
10.1016/j.cell.2007.12.033 .
ISSN
1097-4172 .
PMID
18267078 .
S2CID
15704902 .
External links