Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules

Article abstract of DOI:10.1039/c4ob02522a

Dye-protein fluoromodules consist of fluorogenic dyes and single chain antibody fragments that form brightly fluorescent noncovalent complexes. This report describes two new bichromophoric dyes that extend the range of wavelengths of excitation or emission of existing fluoromodules. In one case, a fluorogenic thiazole orange (TO) was attached to an energy acceptor dye, Cy5. Upon binding to a protein that recognizes TO, red emission due to efficient energy transfer from TO to Cy5 replaces the green emission observed for monochromophoric TO bound to the same protein. Separately, TO was attached to a coumarin that serves as an energy donor. The same green emission is observed for coumarin-TO and TO bound to a protein, but efficient energy transfer allows violet excitation of coumarin-TO, versus longer wavelength, blue excitation of monochromophoric TO. Both bichromophores exhibit low nanomolar K_D values for their respective proteins, >95% energy transfer efficiency and high fluorescence quantum yields.

Full text of DOI:10.1039/c4ob02522a

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Bichromophoric dyes for wavelength shifting of
dye-protein fluoromodules†
Cite this: DOI: 10.1039/c4ob02522a
Ha H. Pham,b,c Christopher Szent-Gyorgyi,c Wendy L. Brotherton,b,c
Brigitte F. Schmidt,c Kimberly J. Zanotti,b,c Alan S. Waggonera,b,c and
Bruce A. Armitage*a,b,c
Dye-protein fluoromodules consist of fluorogenic dyes and single chain antibody fragments that form
brightly fluorescent noncovalent complexes. This report describes two new bichromophoric dyes that
extend the range of wavelengths of excitation or emission of existing fluoromodules. In one case, a
fluorogenic thiazole orange (TO) was attached to an energy acceptor dye, Cy5. Upon binding to a protein
that recognizes TO, red emission due to efficient energy transfer from TO to Cy5 replaces the green
emission observed for monochromophoric TO bound to the same protein. Separately, TO was attached
to a coumarin that serves as an energy donor. The same green emission is observed for coumarin-TO
and TO bound to a protein, but efficient energy transfer allows violet excitation of coumarin-TO, versus
longer wavelength, blue excitation of monochromophoric TO. Both bichromophores exhibit low nano-
molar KD values for their respective proteins, >95% energy transfer efficiency and high fluorescence
quantum yields.
Received 1st December 2014,
Accepted 3rd February 2015
DOI: 10.1039/c4ob02522a
www.rsc.org/obc
small-protein tags.6 Most prominent and frequently used tag-
based strategies include hapten-antibody systems,7 biarsenical
Introduction
Advances in fluorescent protein labeling and imaging have sig- fluorophores FlAsH or ReAsH,8 and various chemoselective
nificantly expanded our understanding of various cellular reactions that exploit specific covalent interactions between
structures and functions. Notably, the development of geneti- synthetic ligands and receptors such as SNAP-tag,9–13 BL-tag,14
cally encodable fluorescent tags sparked by the revolutionary PYP-tag15 and TMP-tag.3,16,17 In addition to the vast potential
discovery of autofluorescent proteins (FPs) has enabled the to finely tune spectral properties via defined chemical modifi-
dynamic tracking of target proteins in living cells.1,2 Although cations, these chemistry-based approaches offer precise
FPs continue to be the most popular and versatile imaging control of protein labeling with high temporal and spatial
tool, their utility in real-time studies is limited by their inher- resolution.6
ently slow maturation times.3 In addition, the interdependence
We recently reported a new chemical tagging platform con-
of spectral and structural properties arising from the covalent sisting of genetically encodable single chain variable fragment
linkage between the fluorophore and protein matrix poses (scFv) antibody proteins and fluorogenic dyes.18 The scFv is
challenges to fine-tuning FPs’ color, brightness and photo- referred to as a fluorescence-activating protein (FAP) and the
stability.4 Furthermore, the current lack of photostable near- combination of a FAP with a dye yields a fluoromodule. Prior
infrared-fluorescing proteins precludes their use for deep to interacting, unbound probes do not fluoresce, resulting in
tissue imaging.5 These limitations have motivated the develop- low background fluorescence signals. Significant fluorescence
ment of alternative technologies that rely on the binding of enhancement occurs when the fluorogenic dye interacts with
exogenous fluorescent dyes to genetically encoded peptide or its associated FAP, whereby the dye is constrained in the
viscous environment of the protein-binding pocket.18 Cognate
FAP–dye pairs are obtained using a library of scFv proteins that
are displayed on the surface of yeast.19 Addition of a fluoro-
aDepartment of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue,
genic dye leads to surface-localized fluorescence from those
yeast that express dye-binding FAPs. Fluorescence activated
cell sorting provides a powerful method for isolating the
desired (but rare) yeast clones from the large excess of yeast
that express nonfunctional scFv proteins. FAPs able to
enhance the fluorescence of our dyes by up to 4 orders of
Pittsburgh, PA 15213, USA. E-mail: army@cmu.edu
bDepartment of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue,
Pittsburgh, PA 15213, USA
cMolecular Biosensor and Imaging Center, Carnegie Mellon University,
4400 Fifth Avenue, Pittsburgh, PA 15213, USA
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob02522a
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magnitude and binders with KD values in the low nanomolar enhances the quantum yield >100-fold, resulting in significant
to high picomolar range have been isolated.18,20
fluorescence in the acceptor channel (Scheme 1, bottom).
Most of our current fluoromodules are based on fluorogenic
cyanine or triphenylmethane monochromophores.18,20–23
Synthesis
These fluorophores typically exhibit relatively small Stokes The bichromophoric dyes and relevant monochromophoric
shifts, meaning that their fluorescence emission spectra are model compounds are shown in Chart 1. Bichromophore DF–A
relatively close to the wavelength of light used to excite the consists of a fluorogenic thiazole orange (TO) donor conju-
dye. This can lead to considerable background due to cellular gated through a flexible diethylene glycol (2p) linker to a Cy5
autofluorescence and light scattering, diminishing the quality acceptor. We refer to DF as TO1 to distinguish it from TO,
of a microscope image. To overcome this generic problem, we which has the same chromophore but a methyl substituent
have coupled FAP technology with Förster resonance energy rather than a propyl sulfonate on the benzothiazole nitrogen.
transfer (FRET) principles to create conditional wavelength- The second bichromophore (D–AF) features a fluorescent cou-
shifting fluoromodules. The “FAP-FRET” approach has been marin donor connected to a fluorogenic thiazole orange accep-
exploited by Bruchez and colleagues to create several multi- tor via a triazole linkage.
chromophore dyes that exhibit (a) amplified fluorescence,24 (b)
The compounds used in this study were synthesized accord-
pH sensitivity25 or (c) color shifting.26 Our aim here is to ing to Scheme 2 and 3. (Full synthetic routes are shown in ESI
extend the scope of FAP-FRET methods based on the use of Schemes S1 and S2†). In synthesizing TO1-2p-Cy5 (Scheme 2),
different fluorogenic donor–acceptor combinations to provide we found that the best coupling strategy involved as the first
new color tags with low fluorescence background, highly step the introduction of the 2p-linker at the TO1-site; this syn-
efficient energy transfer and fluorescence activation, as well as thesis was reported previously.18 The 2p-extended chromo-
high specificity for the tagged protein.
phore exhibits good DMF solubility, in contrast to its parental
chromophore, TO1. The carboxylic acid group of the Cy5 was
activated using TSTU and then reacted with TO1-2p to give the
bichromophoric dye in 60% yield. The coupling yield by this
route is much higher than the alternative, in which a TO1 car-
boxylic acid, which requires an aqueous medium for solubility,
is reacted with Cy5-2p.
A different approach was taken in the synthesis of the cou-
marin-TO bichromophoric dye (D–AF) that employed a shorter
linker and the use of click chemistry (Scheme 3). Coumarin-TO
was synthesized through NHS/DCC coupling of TO-amine with
propiolic acid to give the TO-alkyne, which was then subjected
to standard copper(I) catalyzed azide alkyne cycloaddition con-
ditions27 in the presence of 3-azido-7-hydroxycoumarin28 to
give the coumarin-TO bichromophoric dye in 70% yield. We
also synthesized various monochromophoric dyes (see ESI†),
which were used as model compounds for spectroscopic com-
parisons to the bichromophoric dyes.
Results
Rationale
There are two possible approaches to bichromophoric com-
pounds for use in energy transfer-based wavelength-shifting
fluoromodules. In the first design, a fluorogenic donor (DF) is
covalently linked to an energy acceptor dye (A). Ideally, exci-
tation of DF results in efficient energy transfer and acceptor
fluorescence only when the donor is bound to its cognate FAP
partner due to the low quantum yield of DF in the absence of
the FAP (Scheme 1, top). In contrast, our second bichromo-
phore features an energy donor (D) connected to a fluorogenic
acceptor (AF). Energy transfer can be efficient even in the
absence of a FAP, but the low quantum yield of AF results in
minimal acceptor fluorescence. Binding of a FAP to AF
Spectroscopic characterization of TO1-2p-Cy5 (DF–A)
TO1-2p-Cy5 was designed to function as shown at the top of
Scheme 1, i.e. with donor activation upon binding to a FAP.
The thiazole orange analogue TO1 serves as the light absorber
for wavelengths below 550 nm and subsequently donates
energy to the longer wavelength Cy5 acceptor chromophore.29
The low fluorescence quantum yield (φf) of TO1 in solution30
(2 × 10−4) should lead to weak energy transfer to Cy5 in the
absence of the FAP, whereas binding to an appropriate FAP,
which increases φf for TO1 >1000-fold,18 is expected to yield
greatly enhanced energy transfer to Cy5 in the bichromophoric
construct.
Fig. 1 shows absorption spectra for TO1-2p-Cy5 in aqueous
buffer and MeOH. The ratio of the Cy5:TO1 peaks differs in
the two solvents with the lower value in buffer. In contrast, the
TO1-2p monochromophore shows slightly higher absorbance
in methanol, while the Cy5-2p monochromophore shows a
Scheme 1 Bichromophoric energy transfer dyes combine with FAPs to
generate new fluoromodules.
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Chart 1 Chemical structures of bichromophoric dyes and monochromophoric model compounds.
considerably smaller increase in absorbance in methanol the latter is excited directly or by energy transfer, and that this
versus buffer compared to what is observed in Fig. 1 for the quenching is reasonably efficient in aqueous buffer, presum-
bichromophore (ESI,† Fig. S27). These results indicate that the ably due to the flexible 2p linker allowing the molecule to fold
two chromophores in TO1-2p-Cy5 interact with one another in into a structure that promotes close interaction between the
an aqueous environment. In addition, we measured UV-vis chromophores.
spectra of TO1-2p-Cy5 at 5-fold higher concentration, but the
Excitation of TO1-2p-Cy5 at 480 nm should lead to Cy5
spectral shape did not change significantly (Fig. S28†). This emission in the 650–700 nm range if energy transfer occurs. As
indicates that the perturbations evident in Fig. 1 are due to shown in Fig. 2A, monochromophoric TO1-2p exhibits almost
intramolecular association of the TO1 and Cy5 chromophores, no emission when excited at 480 nm. This is consistent with
rather than intermolecular aggregation.
the low fluorescence quantum yields associated with unsym-
The interaction between the two chromophores is also metrical cyanines due to torsional motions that rapidly deacti-
evident in emission spectra recorded upon direct excitation of vate the excited state.31,32 Observation of substantial Cy5
Cy5. In buffer, the Cy5 emission is approximately four-fold emission when exciting TO1-2p-Cy5 at 480 nm (Fig. 2A, solid
lower in the bichromophore TO1-2p-Cy5 compared with mono- line) indicates that some energy transfer occurs in spite of the
chromophoric Cy5-2p, whereas in methanol, the difference is weakly fluorescent donor TO1. (Excitation of monochromopho-
only 1.2-fold (Fig. S29†). These results indicate that the proxi- ric Cy5-2p at 480 nm does not lead to fluorescence.) This
mity of TO1 in the bichromophore can quench Cy5, whether energy transfer is likely due to the close distance between the
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Scheme 2 Synthesis of bichromophoric TO1-2p-Cy5 (DF–A).
Scheme 3 Synthesis of bichromophoric coumarin-TO (D–AF).
two chromophores, as described above. Interaction (e.g. pi bichromophore exhibited very efficient energy transfer in spite
stacking) between the TO1 donor and Cy5 acceptor could of the anthraquinone donor being nonfluorescent on its own,
result in an enhanced ϕf for TO1, analogous to the higher ϕf although a one-bond linker and appropriate regiochemistry
reported for a dimeric oxazole yellow dye in which the two were required to achieve highly efficient energy transfer.35
chromophores can pi-stack with one another, compared with
The energy transfer that gave rise to Cy5 fluorescence in
the corresponding monomeric version.33 In addition, there are buffer (Fig. 2A) is significantly increased in a viscous solvent
other examples in the literature of energy transfer from weakly such as 90% v/v glycerol/buffer based on the 5-fold higher Cy5
or non-fluorescent donors to covalently attached acceptors.34,35 emission (Fig. 2B). The quantum yield of monochromophoric
In one notable example, an anthraquinone-perylenebisimide Cy5-2p is only enhanced ca. 25% in glycerol/buffer under the
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bands in Fig. 3A indicates that the mono- and bichromophoric
dyes bind to the FAP in similar environments. Nevertheless, as
detailed in Table 1, the KD is ca. 40-fold higher for TO1-2p-Cy5
(12 nM) than for TO1-2p (0.3 nM), indicating a modest degree
of steric and/or electronic repulsion between the protein and
the Cy5 acceptor dye. Nonetheless, the low nanomolar affinity
of HL1.0.1 for TO1-2p-Cy5 is sufficient for the spectroscopy
and imaging experiments described below.
Fig. 3B shows emission spectra (λex = 488 nm) recorded for
TO1-2p-Cy5 and the monochromophoric dye TO1-2p in the
presence of the FAP. As reported previously, fluorescence from
TO1-2p is strongly enhanced by the protein and exhibits an
emission maximum of 536 nm.18 Meanwhile, performing the
same experiment with TO1-2p-Cy5 demonstrates that the TO1
fluorescence is strongly quenched, but is replaced by sensi-
tized Cy5 fluorescence at 668 nm due to energy transfer. The
efficiency of energy transfer, based on the extent of TO1
quenching, is 96%. The Cy5 emission maximum is also red-
shifted relative to the spectrum recorded in buffer with direct
excitation of Cy5, indicating that Cy5 is not extended far away
from the protein but rather is in close proximity to the FAP, as
suggested by the higher KD value for TO1-2p-Cy5 relative to
TO1-2p (Table 1).
Fig. 1 UV-vis absorbance spectra of 1.0 μM TO1-2p-Cy5 in aqueous
buffer and MeOH.
same conditions (Fig. S30†), so the large enhancement
observed for the bichromophore is attributed to the higher
quantum yield of the TO1 donor (compare dashed lines in
Fig. 2A and 2B). In addition, there is reduced quenching of
Cy5 by TO1, as indicated by 5-fold greater fluorescence upon
direct excitation of Cy5 in glycerol versus buffer (Fig. S30†),
consistent with the findings for methanol.
The utility of TO1-2p-Cy5 as a wavelength-shifting fluorogen
was next demonstrated in the context of protein-based fluoro-
modules. The bichromophore is activated by the FAP HL1.0.1-
TO1, an scFv antibody fragment previously selected for
binding to TO1-2p.18 The similarity of the TO1 absorption
Confocal microscopy
The net effect of using TO1-2p-Cy5 is to shift the emission
wavelength of the TO1-based fluoromodule by ca. 130 nm to
Fig. 2 Fluorescence emission (λex = 480 nm) spectra for TO1-2p and TO1-2p-Cy5 in aqueous solution (A) and in 90% v/v glycerol/water (B).
Fig. 3 (A) Absorbance spectra of 1.0 μM TO1-2p or TO1-2p-Cy5 in the presence of 1.0 μM FAP HL1.0.1-TO1. (B) Fluorescence emission spectra
recorded for 200 nM TO1-2p and TO1-2p-Cy5 in the presence of 2 µM FAP HL1.0.1-TO1. Samples were excited at 488 nm.
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affect the binding of certain dye-FAP pairs (vide infra). Fig. 5
shows absorption spectra for the bichromophoric coumarin-
TO compound in aqueous buffer and MeOH. While the posi-
tion of the absorption peak of TO is similar in the two sol-
vents, the coumarin absorption band noticeably red-shifts
from 350 to 420 nm in aqueous buffer. A similar trend was
observed in the absorption spectrum of the monochromopho-
ric coumarin (Fig. S31†). These bathochromic shifts are
likely due to deprotonation of the aromatic alcohol in the
coumarin unit in PBS solution (pH = 7.4), consistent with pre-
vious reports on the red-shifted absorption of deprotonated
7-hydroxycoumarin in comparison to the neutral molecular
species.36
We next studied the fluorescence properties of free cou-
marin-TO in an aqueous environment (Fig. 6). Direct excitation
of the coumarin monochromophore at 370 nm yields the
expected emission band that peaks at 470 nm. On the other
hand, bichromophoric coumarin-TO exhibits almost no fluo-
rescence when excited at the same wavelength. The lack of
fluorescence signal is expected since, in the absence of a TO-
binding FAP, the TO acceptor can readily quench the excited
coumarin, but then nonradiatively dissipates energy rather
than fluorescing. Moreover, the nearly complete quenching of
coumarin’s emission suggests a highly efficient (>95%) energy
transfer from the donor to the acceptor even in the absence of
a cognate FAP.
Table 1 Properties of bichromophores TO1-2p-Cy5 and coumarin-TO
TO1-2p TO1-2p-Cy5
TO
Coumarin-TO
ε (M-1 cm−1 a
)
49 500
504
49 500 (TO1)
200 000 (Cy5)
511 (TO1)
650 (Cy5)
12
0.37
96%
63 000 17 300 (coumarin)
49 200 (TO)
λabs (nm)b
505
400 (coumarin)
508 (TO)
1.1
KD (nM)b
Φb
0.3
0.47
N/A
0.17
0.89
N/A
0.87
FRET%b
99%
a Determined in MeOH. b Determined in the presence of cognate FAP.
the red as shown in Fig. 3B. The large spectral shift is useful
for confocal microscopy, where images of yeast that display the
HL1.0.1-TO1 FAP on the cell surface were stained with the cell-
impermeant TO1-2p or TO1-2p-Cy5 dyes (Fig. 4). Staining with
the monochromophoric reagent TO1-2p yields fluorescence
only in the green channel. In contrast, TO1-2p-Cy5 yields very
weak TO1 fluorescence in the green channel but strong red
channel fluorescence when Cy5 is directly excited or when TO1
is excited, consistent with the efficient energy transfer
observed spectroscopically. Quantitative analysis of the images
demonstrates the improved signal-to-noise obtained from
staining with the bichromophoric reagent (right panel, Fig. 4).
Spectroscopic characterization of coumarin-TO (D–AF)
Chart 1 shows the structure of our second bichromophore,
Interestingly, in 90% glycerol, where TO fluorescence
coumarin-TO, along with the parental coumarin and TO con- should be activated by the viscous solvent, we observe a four-
trols. As noted above, TO differs from TO1 in the nature of sub- fold increase in fluorescence from bichromophoric coumarin-
stituents attached to the benzothiazole heterocycle (methyl vs. TO versus the monochromophore TO (Fig. S32†). This large
propylsulfonate for TO and TO1, respectively). Although this increase occurs in spite of a 30% decrease in absorbance for
structural change is remote from the fluorophore, it might the bichromophore. These results suggest that, similar to TO1-
Fig. 4 Visualization of energy transfer using yeast surface-displayed HL1.0.1-TO1 FAP. Yeast expressing FAP on the cell surface were excited at
488 nm (TO1 Ex) or 633 nm (Cy5 Ex) and visualized by laser confocal microscopy using either 100 nM TO1-2p or bichromophore TO1-2p-Cy5.
Shown left to right are donor (500–550 nm BP filter), acceptor (650–710 nm BP), and FRET (650–710 nm BP) channels. At the far right are shown
scan profiles of these channels through cells along 50 µm transects (horizontal axis), traced in yellow with open circles marking transect origins. Ver-
tical axes plot per pixel signal represented as proportion of 12-bit intensity scale. Solid ovals mark positions of fluorescent cells and dotted ovals
mark positions of dark cells that do not express FAP due to absence of encoding plasmid. Control images that localize dark cells and microscopy
parameters are shown in Fig. S26 and Table S2.†
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R1, a recombinant scFv originally selected for a photostable
derivative of TO21 was used for these experiments based on
its ability to bind and activate TO with subnanomolar affinity
(174 pM) and strongly enhanced fluorescence quantum yield
(ϕf = 0.89, a 4400-fold increase) (Table 1). Interestingly, R1
showed minimal activation of TO1 (Fig. S33†), further illustrat-
ing the sensitivity of these proteins toward the nature of the
substituent on the benzothiazole heterocycle of thiazole orange
dyes.
Fig. 7 compares the absorption and emission spectra of
coumarin-TO in the absence and presence of R1. In the pres-
ence of the FAP, the TO absorption band becomes markedly
sharpened with a defined vibronic shoulder at 475 nm. A
similar effect was observed for monochromophoric TO
(Fig. S34†). These results confirmed that the TO moiety was
tightly constrained within the binding pocket of the FAP, reduc-
ing the conformational freedom that contributes to spectral
broadening. Similar to TO1-2p-Cy5 with its cognate FAP, a
modest reduction in affinity was observed for coumarin-TO
relative to TO (KD = 1.1 nM versus 174 pM, Table 1), again
suggesting that steric hindrance arising from the close proxi-
mity of the donor and acceptor affects the binding of the
bichromophoric dyes to their respective FAPs, although
the relatively high affinities for the monochromophores pro-
vides considerable tolerance for binding of bichromophoric
derivatives.
Fig. 5 UV-Vis absorbance spectra for coumarin-TO in aqueous buffer
and MeOH.
Fluorescence emission spectra of coumarin, TO and cou-
marin-TO in the presence of excess R1 were recorded using
400 nm (coumarin) excitation (Fig. 7B). The efficiency of
energy transfer from coumarin to TO, based on the decrease in
the donor emission peak, is ca. 99% and leads to strong, sensi-
tized TO emission. (Excitation of TO alone at 400 nm leads to
very weak emission.)
Fig. 6 Fluorescence emission spectra (λex = 370 nm) for coumarin, TO
and coumarin-TO in aqueous solution.
2p-Cy5, some degree of interaction occurs between the two
chromophores in coumarin-TO. However, unlike the DF–A
bichromophore TO1-2p-Cy5, where interaction leads to
quenching of the Cy5 acceptor, in the D–AF bichromophore
coumarin-TO, interaction leads to significantly enhanced fluo-
rescence from the TO acceptor.
Confocal microscopy
As illustrated in Fig. 8, yeast cells expressing the R1 protein on
their surface were imaged with either 10 nM coumarin-TO or
The photophysical properties of coumarin-TO in the pres-
ence of a FAP were next examined. TO gave weaker fluo- monochromophoric TO control. As expected, direct excitation
rescence in the presence of the protein HL1.0.1-TO1, of TO using a 488 nm laser leads to bright fluorescence signals
compared with the propylsulfonate-substituted TO1 (Fig. S33†),
in both stained samples (center column). When excited at
so we used a different FAP for these experiments. Specifically, 405 nm, no signal is observed in the 420–480 nm range due to
Fig. 7 (A) Absorption spectra of TO and coumarin-TO in the presence of R1 FAP. (B) Fluorescence emission spectra (λex = 400 nm) of coumarin, TO
and coumarin-TO in the presence of R1.
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Fig. 8 Visualization of energy transfer using yeast surface-displayed R1 FAP. Confocal microscopy imaging was carried out in the presence of
10 nM TO control (top) or bichromophore Coumarin-TO (bottom). Samples were excited using either 405 nm (Coumarin Ex) or 488 nm (TO Ex)
lasers. Three different channels were examined: donor channel (420–480 nm BP filter, left), acceptor channel (505–550 nm BP, middle) and FRET
channel (505–550 nm BP, right). At the far right are shown scan profiles of these channels through cells along 50 µm transects (horizontal axis),
traced in yellow with open circles marking transect origins. Vertical axes plot per pixel signal represented as proportion of 16-bit intensity scale.
Solid ovals mark positions of fluorescent cells and dotted ovals mark positions of dark cells that do not express FAP due to absence of encoding
plasmid. Control images that localize dark cells and other microscopy parameters are shown in Fig. S26 and Table S2.†
(a) lack of absorbance by monochromophoric TO and (b) a class of fluorescent labels that overcome some of these limit-
efficient energy transfer for coumarin-TO. When the samples ations. The use of fluorogenic dyes, which become fluorescent
were excited at 405 nm (coumarin) and emission monitored at only after binding to the cognate FAP, not only lowers the fluo-
505–550 nm (TO), the bichromophoric dye exhibited strong rescence background, but also reduces the labeling time by
staining of the yeast surface (lower right image), reflecting the obviating the need to remove excess probes. As a result, we are
efficient energy transfer.
able to visualize cell surface labeling immediately after the
addition of appropriate bichromophores without washing
(Fig. 4 and 8). The use of FRET-capable bichromophores pro-
vides the opportunity to further improve signal-to-noise, due
to the red-shifting of the emission relative to simpler mono-
chromophoric dyes. The sharp contrast between non-expres-
sing and FAP-expressing yeast cells (Fig. 4, 8 and S26†)
confirms the lack of fluorescence background in the absence
of target protein. In addition, the high FRET efficiency (>95%)
achieved in our bichromophoric systems leads to low signal
observed in both control donor channels.
Although the results validate the strategy depicted in
Scheme 1, the reality is somewhat more complicated. For
example, the spectral data indicate that the TO1 and Cy5 chro-
mophores interact, based on perturbation of the UV-vis spectra
and quenching of Cy5 fluorescence in the bichromophore.
Similarly, the 4-fold higher fluorescence of TO when linked to
coumarin, even when directly exciting TO, indicates interaction
between the two chromophores in coumarin-TO. Thus, it is
likely that the bichromophore molecules adopt a folded con-
formation in water, bringing the two chromophores into close
proximity before unfolding in order to allow the TO or TO1
fluorogen to bind to the FAP (Scheme 4).
Discussion
The ability to track target proteins in real time is fundamental
to understanding dynamic events in living cells.6 Although
fluorescent protein technology remains one of the most power-
ful and commonly used tools in cell biology, the inherently
slow maturation time due to the post-translational chemical
reactions needed to form the fluorophore as well as the
susceptibility to oligomerization places limitations on the use
of FPs in real-time imaging.3 When compared to synthetic
probes that utilize the best organic fluorophores, FPs generally
have lower brightness and photostability (collectively known as
photon output), leaving room for improvement for experiments
that require single-molecule resolution and/or high sensitivity.
On the other hand, the key problem in labeling proteins
using chemical tags arises from the high fluorescence back-
ground of the unbound probes. Thus, extensive washing prior
to imaging is often required to improve signal-to-noise ratio37
but still may not result in the complete elimination of non-
specific binding.38,39 More importantly, the necessity of a
wash-out step prevents rapid and continuous detection of
target protein, hence defeating the purpose of monitoring bio-
molecules in real time.
It should be noted that the concept of fluorogenic probes
has been previously exploited for live-cell protein imaging.
Notable systems include the tetracysteine biarsenicals FlAsH
and ReAsH,1,8 the intramolecular association PYP tag,15 the
FRET-quenched SNAP-tags,11–13 the BL-tag14 and most recently
The fluorogenic bichromophores and genetically encodable
FAPs described above represent a strategy to rationally design
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For example, conjugation of 1, 2 or 4 Cy3 donor dyes to a MG
fluorogen yields dendritic (DF)n–A “dyedrons” that exhibit
strong red emission when the MG fluorogen is bound to a
cognate FAP.24 Moreover, the presence of multiple Cy3 donors
leads to amplified emission due to the higher absorbance
cross section at donor wavelengths. In a second example,
linking MG to tetramethylrhodamine yields D–AF bichromo-
phores with efficient FRET and red emission.26 The authors
also reported an interesting feature: the equilibrium between
nonfluorescent carbinol and fluorogenic triphenylmethane
forms of MG depends strongly on the nature of the linkage
between the two chromophores, allowing tuning of the emis-
sion between yellow-orange (TMR) and red (MG).
The TO1-2p-Cy5 bichromophore reported here differs struc-
turally from the previously reported conjugate by the presence
of the 2p linker, which adds 14 atoms between the TO1 and
Cy5 fluorophores. The extended linker length could contribute
to the 5-fold higher affinity for the FAP that we observe relative
to the pH-dependent TO1-Cy5 bichromophore binding to the
same FAP. In addition, the quantum yield for sensitized fluo-
rescence is 6-fold higher in the case of TO1-2p-Cy5 at neutral
pH compared with the pH-dependent TO1-Cy5 under acidic
conditions. This is due at least in part to the more efficient
energy transfer observed in the present case.
Our work with coumarin-TO extends the range of FRET
bichromophores to blue excitation with green emission. The
exceptional affinity of TO for the new FAP R1 (KD = 0.17 nM)
and high quantum yield (ϕf = 0.89) provide an excellent start-
ing point from which to create a bichromophoric analogue.
The coumarin-TO bichromophore retains reasonably high
affinity for R1 (KD = 1.1 nM), while maintaining a high
quantum yield (ϕf = 0.87) due to the nearly quantitative FRET
from coumarin to TO. If subnanomolar affinity were needed, it
is possible that a longer linker between the two chromophores
would restore some of the higher affinity observed for the TO
monochromophore; however, this might also reduce the FRET
efficiency. Alternatively, affinity maturation of R1 using the
bichromophore could lead to mutants with lower KD values for
coumarin-TO.
Scheme 4 Folded conformation of bichromophore leads to moderate
FRET prior to FAP binding. (An analogous scheme could be drawn for
the D–AF bichromophore coumarin-TO).
the fluorogenic split inteins.4 Aside from the acute toxicity of
arsenic, tetracysteine tags display non-specific affinity for iso-
lated thiols,5 which counter-productively necessitates a lengthy
washout.38 Moreover, ReAsH is known to generate reactive
singlet oxygen species, contributing to the overall photo-
instability of the examined system. Drawbacks of other FRET-
based quenching approaches result from the slow kinetics of
covalent interactions between the fluorogens and protein tags.
This typically requires high probe concentration (e.g. at least
500 nM for BL-tag) and lengthy incubation time6,37 relative to
our immediate fluorescence response with low nanomolar con-
centrations of bichromophores. Further, the irreversibility of
covalent bond formation in other fluoromodule technologies
prevents the exchange of photobleached probes with fresh
ones, a mechanism that has been shown to improve the photo-
stabilty of FAP-based systems.22 Above all, with thousand-fold
fluorescence enhancements and high FRET efficiency, our
FAP-FRET design compares favorably to the fluorogenic SNAP-
tag (<100-fold)12,13 and PYP-tag (20-fold).15 A recent attempt to
improve fluorescence signal for SNAP-tag technology results in
ca. 300-fold activation via the use of a FAPL probe.11
The different binding affinities and emission colors of TO1-
2p-Cy5 and TO1-2p in complex with HL1.0.1-TO1 FAP has
recently been exploited to develop a quantitative pulse-chase
flow cytometric screening technology that monitors in real
time the internalization of a GPCR receptor.40 In this protocol,
the FAP is fused to an extracellular domain of the ADRB2
receptor expressed on the surface of a mammalian cell. A
pulse using TO1-2p-Cy5 enables one to detect the uptake of
receptor into endocytic vesicles upon agonist stimulation, fol-
lowed by a chase with TO1-2p that labels receptor remaining
on the surface. This method was used to follow receptor desen-
sitization and resensitization.
Conclusions
The results presented here demonstrate new strategies for
changing the color of protein-dye fluoromodules: the fluoro-
gen used to select complementary FAPs from the scFv library
can be subsequently conjugated to an appropriate FRET donor
or acceptor to obtain new fluoromodules with different exci-
tation (D–AF) or emission (DF–A) wavelengths. This comple-
ments prior work in which promiscuous FAPs could be used
to generate a range of colors.20,23,41 The modularity of the tech-
nology and tolerance of the FAPs to conjugating a second dye
to the original fluorogen allows these new fluoromodules to be
used without necessarily requiring new proteins to be selected,
although subsequent affinity maturation might be useful for
enhancing affinity. In principle, different fluorogenic donor–
In other work, TO1 was conjugated to a pH-dependent
version of Cy5, yielding a DF–A bichromophore that exhibits
green emission from TO1 when bound to an appropriate FAP
at neutral or basic pH (where the Cy5 is deprotonated and
therefore inactive as an energy acceptor), but red emission at
acidic pH, where Cy5 protonation promotes FRET. This prop-
erty was used to monitor endocytic cell uptake of FAP-protein
fusions.25
Bruchez and colleagues have documented the effectiveness
of FAP-FRET systems based on FAP activation of fluorogenic
malachite green acceptors for imaging proteins in live cells.
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acceptor bichromophores can be created to provide a catalogue
Ligand depletion assay to determine KD. Binding affinity of
of colors that can be used for simultaneous tracking of mul- TO1-2p-Cy5 and coumarin-TO to their cognate FAPs were deter-
tiple fusion proteins through multicolor imaging/detection or mined using 20 nM HL1.0.1-TO1 and 15 nM R1, respectively.
temporally resolved imaging/detection of a single fusion Corresponding KD values for monochromophores TO1-2p and
protein through labeling at different times with mono- or TO were also measured using 2 nM HL1.0.1-TO1 and 1 nM R1,
bichromophoric reagents.
respectively. In the case of bichromophores, samples were
excited at the donor absorption wavelength and fluorescence
signal of acceptor fluorophores Cy5 and TO were monitored.
Fluorescence of each FAP–dye complex was corrected by sub-
tracting the fluorescence of a dye only solution. All KD values
were extrapolated from the non-linear regression using Graph-
pad Prism 5.0 and a ligand depletion algorithm43
Experimental section
Full synthetic details and characterization data for the bi- and
mono-chromophoric dyes are provided in the ESI† (Schemes
S1-S2, Table S1 and Fig. S1–S24).
2
1=2
X þ KD ꢀ R ꢀ ½ðꢀX ꢀ KD ꢀ RÞ ꢀ 4XRꢁ
Y ¼
Protein purification
2
TO1-binding FAP HL1.0.1-TO1 is an affinity-matured version of
a FAP that was originally selected to bind to TO1-2p.18 New
FAP R1 emerged as one of the potential binders of αCN-TO, a
photostable TO analogue.21 Sequence data and purity assess-
ment for the R1 FAP are provided in ESI† (Fig. S25) and com-
plete details regarding the selection of R1 from a yeast surface-
displayed library will be described elsewhere.
The pPNL6 plasmid containing the R1 clone was extracted
using a Zymoprep I kit (Zymo Research). This plasmid was
then transformed into Mach1-T1R E. coli. Sequencing was per-
formed by GeneWiz. Expression of the soluble R1 protein in
the Mach1-T1 strain was accomplished as described pre-
viously.20 HL1.0.1-TO1 protein was secreted from yeast as
described.18
where X is the concentration of dye and R is the concentration
of FAP–dye complex at the maximal fluorescence plateau.
Determination of fluorescence quantum yields. Quantum
yields for TO and TO1-2p were determined in the presence of
excess FAP R1 and HL1.0.1-TO1, respectively. Fluorescein in
0.1 M NaOH (Φf = 0.925 at 22 °C) and Lucifer Yellow in water
(Φf = 0.21 at 20 °C) were used as quantum yield standards
because of the spectral overlap with the examined fluorogens.
Quantum yields were determined from FAP–dye complexes
having an absorbance between 0.02 and 0.1 at and above λmax
.
The fluorescence spectrum of each sample was integrated
using Origin 6.1 and plotted against its absorbance using a
linear regression fit to give slope M. Quantum yields (Φx) were
determined using the following equation:
ꢀ
ꢁ ꢀ
ꢁ
Buffer
2
Mx
MSD
ηx
Φx ¼ ΦSD
All experiments were conducted in a modified phosphate
buffered saline system (PBS+). The buffer contained 137 mM
NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 2 mM
EDTA and 0.1% w/v Pluronic F-127. Buffer was adjusted to
pH = 7.4.
ηSD
where Φsd is the quantum yield of the standard dye, Mx and
Msd are the slopes derived from the plot described above, and
ηx and ηSD are the refractive indices of the PBS+ buffer and
standard solvent, respectively. Refractive indices of 1.333 and
1.334 were used for water (also PBS+ buffer) and 0.1 M NaOH.
Quantum yields of FAP–bichromophore complexes (ΦC)
were calculated by the following equation:
Optical spectroscopy
UV-Vis spectra were recorded with baseline correction on a
Cary 300 Bio UV-Visible spectrophotometer at 20 °C. For fluoro-
module-containing samples, buffer was blanked in the presence
of the FAP. Corrected fluorescence spectra of HL1.0.1-TO1
FAP complexes were taken at RT on a Quantamaster mono-
chromator fluorimeter (Photon Technology International); all
other fluorescence spectra were measured using a Cary Eclipse
fluorescence spectrophotometer at 20 °C.
ΦC ¼ ΦA ꢂ E
where ΦA is the quantum yield of the acceptor chromophore
and E is the energy transfer efficiency, estimated from the
observed quenching of the donor fluorescence in the presence
of the acceptor dye.
Fluorescence microscopy. Confocal fluorescence of yeast
surface-displayed R1 and HL1.0.1-TO1 in the presence of
Determination of KD values for soluble FAP42
Range-finding experiment. Range finding experiments were corresponding monochromophores and bichromophores were
performed to determine approximate KD and binding satur- imaged on a Carl Zeiss LSM 510 Meta Confocal Microscope
ation level for subsequent ligand depletion assay. A serial using live cells immobilized on Concanavalin-coated Mattek
dilution of 10 : 1 FAP : dye solution starting with 1 μM FAP was dishes. For R1, separate samples containing 10 nM TO and 10
prepared along with a control dye : buffer dilution. All solu- nM coumarin-TO were excited using either a 405 nm or
tions were incubated overnight at room temperature. Fluo- 480 nm laser line. A 420–480 nm band-pass (BP) filter was
rescence signals were recorded the following day using Tecan used to detect coumarin signal whereas TO fluorescence was
Infinite M1000 Plate Spectrometer.
monitored using a BP 505–550 nm filter. For yeast-expressing
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Paper
HL1.0.1-TO1 FAP, separate samples containing 100 nM TO1-2p 14 S. Mizukami, S. Watanabe, Y. Akimoto and K. Kikuchi,
and 100 nM TO1-2p-Cy5 were excited using 488 nm (8% J. Am. Chem. Soc., 2012, 134, 1623–1629.
power) or 633 nm (10% power) laser lines. TO1-2p and Cy5 15 Y. Hori, H. Ueno, S. Mizukami and K. Kikuchi, J. Am. Chem.
signals were respectively monitored using a BP 505–550 nm Soc., 2009, 131, 16610–16611.
filter or a BP 650–710 nm filter. Images from all channels were 16 L. W. Miller, Y. Cai, M. P. Sheetz and V. W. Cornish, Nat.
adjusted in Zeiss ZEN 2009 software using identical settings Methods, 2005, 2, 255–257.
that display the full linear signal range. Scan profiles along 17 N. T. Calloway, M. Choob, A. Sanz, M. P. Sheetz,
transects in Fig. 4 and 8 were respectively carried out in
ImageJ 2.0 software on unaltered 12-bit and 16-bit LSM files.
L. W. Milller and V. W. Cornish, ChemBioChem, 2007, 8,
767–774.
18 C. Szent-Gyorgyi, B. F. Schmidt, Y. Creeger, G. W. Fisher,
K. L. Zakel, S. Adler, J. A. Fitzpatrick, C. A. Woolford,
Q. Yan, K. V. Vasilev, P. B. Berget, M. P. Bruchez,
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235–240.
19 M. J. Feldhaus, R. W. Siegel, L. K. Opresko, J. R. Coleman,
J. M. Feldhaus, Y. A. Yeung, J. R. Cochran, P. Heinzelman,
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20 H. Özhalici-Ünal, C. Lee Pow, S. A. Marks, L. D. Jesper,
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Acknowledgements
K.J.Z. gratefully acknowledges support from a DoD, Air Force
Office of Scientific Research, NDSEG Fellowship 32 CFR 168a.
This work is supported by the U.S. National Institutes of
Health (grant U54 RR022241). NMR instrumentation at CMU
was partially supported by NSF (CHE-0130903). Mass spec-
trometers were funded by NSF (DBI-9729351). We are grateful
to Prof. Dane Wittrup (MIT) for providing an aliquot of the
yeast scFv surface-displayed library.
21 N. I. Shank, H. H. Pham, A. S. Waggoner and
B. A. Armitage, J. Am. Chem. Soc., 2013, 135, 242–251.
22 N. I. Shank, K. J. Zanotti, F. Lanni, P. B. Berget,
A. S. Waggoner and B. A. Armitage, J. Am. Chem. Soc., 2009,
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