A stilbene that binds selectively to transthyretin in cells and remains dark until it undergoes a chemoselective reaction to create a bright blue fluorescent conjugate

Article abstract of DOI:10.1021/ja104999v

We describe a non-fluorescent, second generation stilbene that very selectively binds to transthyretin in complex biological environments and remains dark until it chemoselectively reacts with the pK_a-perturbed Lys-15 ε-amino group of transthyr

Full text of DOI:10.1021/ja104999v

Published on Web 10/21/2010
A Stilbene That Binds Selectively to Transthyretin in Cells
and Remains Dark until It Undergoes a Chemoselective
Reaction To Create a Bright Blue Fluorescent Conjugate
Sungwook Choi,† Derrick Sek Tong Ong,‡ and Jeffery W. Kelly*,‡,§
Department of New Drug DiscoVery and DeVelopment, Chungnam National UniVersity,
Daejon, 305-764, Republic of Korea, and Departments of Chemistry and Molecular and
Experimental Medicine, and The Skaggs Institute for Chemical Biology, The Scripps Research
Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
Received June 8, 2010; E-mail: jkelly@scripps.edu
Abstract: We describe a non-fluorescent, second generation stilbene that very selectively binds to
transthyretin in complex biological environments and remains dark until it chemoselectively reacts with the
pKa-perturbed Lys-15 ε-amino group of transthyretin to form a bright blue fluorescent conjugate. Stilbene
A2 is mechanistically unusual in that it remains non-fluorescent in cell lysates lacking transthyretin, even
though there is likely some proteome binding. Thus, it is especially useful for cellular imaging, as background
fluorescence is undetectable until A2 reacts with transthyretin. The mechanistic basis for the effective lack
of environment-sensitive fluorescence of A2 when bound to, but before reacting with, transthyretin is
reported. Stilbene A2 exhibits sufficiently rapid transthyretin conjugation kinetics at 37 °C to enable
pulse-chase experiments to be performed, in this case demonstrating that transthyretin is secreted from
HeLa cells. As the chase compound, we employed C1, a cell-permeable, highly selective, non-covalent,
transthyretin-binding dihydrostilbene that cannot become fluorescent. The progress reported is viewed as
a first and necessary step toward our long-term goal of creating a one-chain, one-binding-site transthyretin
tag, whose fluorescence can be regulated by adding A2 or an analogous molecule. Fusing proteins of
interest to a one-chain, one-binding-site transthyretin tag regulated by A2 should be useful for studying
folding, trafficking, and degradation in the cellular secretory pathway, utilizing pulse-chase experiments.
Immediate applications of A2 include utilizing its conjugate fluorescence to quantify transthyretin
concentration in human plasma, reflecting nutritional status, and determining the binding stoichiometry of
kinetic stabilizer drugs to transthyretin in plasma.
of wavelengths;10 thus, mutagenesis has proven to be a very
useful approach to alter the photophysical properties of the
Introduction
chromophore.1 In a complementary approach, a tetra-cysteine tag
on a protein of interest can be used to bind the As-containing latent
fluorophores FlAsH or ReAsH, rendering the conjugate fluorescent
through a sulfide exchange reaction that can be conformation- or
quaternary structure-dependent.11-14 Additional methodology to
fluorescently tag a protein of interest includes the enzymatic
conjugation of fluorophores to proteins,15-18 as well as the
snap19-21 and halo tag22,23 approaches.
The development of fluorescent tags and fluorescent biosen-
sors to image processes within living cells with spatial resolution
has transformed what can be accomplished in biological
research.1-3 A very useful macromolecule for fluorescence
tagging and a component of many biosensors is the green
fluorescent protein (GFP), which first folds and then undergoes
an autocatalytic intramolecular chemical reaction to form its
chromophore.4-9 Mutants of GFP exhibit emission at a spectrum
Wild-type (WT) transthyretin is a tetrameric human plasma
protein composed of identical 127-amino-acid ꢀ-sheet-rich
† Department of New Drug Discovery and Development, Chungnam
National University.
‡ Department of Chemistry and The Skaggs Institute for Chemical
Biology, The Scripps Research Institute.
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Cormier, M. J. Gene 1992, 111, 229–233.
§ Department of Molecular and Experimental Medicine, The Scripps
Research Institute.
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59, 223–239.
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Figure 1. (A) Crystal structure of the TTR-(thyroxine)2 complex with differentially colored subunits. (B) Structures of covalent TTR modifiers A1 and A2
and the fluorescent propylamide stilbene analogue B1 that is not capable of reacting with WT-TTR. (C) Depiction of the structure of the two TTR thyroxine
binding sites undergoing a reaction with A1 or A2 to create a WT-TTR-(stilbene)2 conjugate.
subunits (Figure 1A).24-27 The more labile dimer-dimer
interface of transthyretin (TTR) (bisected by the crystallographic
two-fold axis; Figure 1A) creates two identical funnel-shaped
thyroxine (T4) binding sites that are interconverted by a C2 axis
perpendicular to the crystallographic two-fold axis.27-29 Previ-
ously, we reported stilbenes that bind to the T4 binding sites
within TTR in human plasma with very high selectivity over
the other 4000+ proteins present.30,31 Active ester analogues
of these stilbenes then chemoselectively react with the pKa
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A Stilbene Fluorescent Tag for Transthyretin
A R T I C L E S
mm. The samples were excited at 328 nm, and the emission spectra
were collected from 350 to 550 nm.
perturbed lysine-15 (K15) ε-amino group of TTR to form the
amide-linked stilbene conjugates.32 An X-ray crystal structure
defined the binding orientation of the stilbene substructure and
the apparent cis-amide linkage to TTR.32
Kinetics of the A2-TTR Conjugation Reaction. The covalent
TTR modifier A2 (1 µL of 0.6 mM solution in DMSO, final
concentration 6 µM) was added to 90 µL of HeLa cell lysate (final
protein concentration 2 µg/µL) and 10 µL of WT-TTR (20 µM in
pH 7.0 phosphate buffer, final concentration 2 µM). The time-
dependent fluorescence change of the conjugate derived from A2
was monitored utilizing a microplate spectrophotometer reader
(Gemini SpectraMax, Molecular Devices, Sunnyvale, CA) for 3 h
at 37 °C. The fluorescence (λex ) 328 nm and λem ) 430 nm) was
measured from the bottom of the plate without shaking.
Herein, we describe a non-fluorescent, second generation
stilbene active ester, A2 (Figure 1B), and related stilbenes that
selectively bind to and then chemoselectively react with the TTR
protein in complex biological environments, creating blue
fluorescent conjugates as a consequence of amide bond forma-
tion (Figure 1C). It is atypical that stilbenes remain dark when
bound to proteins, and in that regard stilbene A2 is especially
useful for cellular imaging. Stilbene A2 (Figure 1B) also exhibits
highly selective binding to TTR within the secretory pathway
of eukaryotic cells and then undergoes a chemoselective amide-
bond-forming reaction that renders the conjugate fluorescent.
Stilbene A2 exhibits sufficiently rapid transthyretin conjugation
kinetics at 37 °C to enable pulse-chase experiments to be
performed, the chase utilizing a highly selective, non-covalent
TTR binder that cannot become fluorescent.
The progress reported is viewed as a first and necessary step
toward the creation of a one-chain, one-binding-site TTR tag
whose fluorescence can be turned on when a small molecule of
the type described herein is added to cells to form a TTR-
stilbene conjugate. Folded, monomeric versions of TTR have
been published33,34 and were created by introducing sterically
demanding mutations into both of TTR’s quaternary structural
interfaces.28,35 We have already linked subunits a and c (Figure
1A), employing a low complexity linker using established
molecular biological approaches.28 We are currently seeking
mutations that prevent the one-chain, one-binding-site TTR
molecule from dimerizing to form the equivalent of the tetramer.
Fusing proteins of interest to a one-chain, one-binding-site
transthyretin tag regulated by A2 should be especially useful
for following protein maturation and degradation in the secretory
pathway of eukaryotic cells.36-38
Binding Stoichiometry of A2 to WT- and K15A-TTR. To 1
mL of WT-TTR (5 µM) in HeLa cell lysate (2 µg/µL total protein
concentration, excluding TTR) or K15A-TTR (5 µM) in pH 7.0
phosphate buffer in a 2 mL Eppendorf tube was added A2 (5 µL
of a 2 mM solution in DMSO, final concentration 10 µM), and
then the solution was incubated at 37 °C for 24 h on a rocker plate
(30 rpm). A 1:1 (v/v) slurry of unfunctionalized sepharose resin in
10 mM Tris, 140 mM NaCl, pH 8.0 (TSA) buffer was added, and
the solution was incubated for 1 h at 4 °C on a rocker plate (18
rpm). The solution was then centrifuged, and the supernatant was
divided into two 400 µL aliquots, which were added to 200 µL of
a 1:1 (v/v) slurry of anti-TTR antibody-conjugated sepharose resin
in TSA. The solution was gently rocked (18 rpm) at 4 °C for 20
min and then centrifuged, and the supernatant was removed. The
resin was washed three times by shaking for 1 min with 1 mL of
TSA. After centrifugation to remove the supernatant, 155 µL of
triethylamine (100 mM, pH 11.5) was added to the sepharose resin
to dissociate the TTR and bound test compound from the resin,
and the suspension was vortexed for 1 min. After centrifugation,
the supernatant was analyzed by reverse-phase HPLC on a Waters
600 E multisolvent delivery system, using a Waters 486 tunable
absorbance detector, a 717 autosampler, and a ThermoHypersil
Keystone Betabasic-18 column (150 Å pore size, 3 µm particle
size). The “A” mobile phase comprises 0.1% TFA in 94.9% H2O
+ 5% CH3CN, and the “B” mobile phase is made up of 0.1% TFA
in 94.9% CH3CN + 5% H2O. Linear gradients were run from 100:0
A:B to 0:100 A:B for 30 min.
Quantum Yield Measurement. The covalent TTR modifier A1
or A2 or the non-covalent TTR ligand B1 (15 µL of a 1.44 mM
solution in DMSO, final concentration 7.2 µM) was added to 3
mL of a solution of WT-TTR homotetramer (0.2 mg/mL, 3.6 µM)
in 10 mM phosphate, 100 mM KCl, and 1 mM EDTA (pH 7.0
phosphate buffer). The samples were vortexed and incubated for
18 h at 25 °C. Quantum yields were measured by following the
instructions at www.jobinyvon.com/usadivisions/Fluorescence/ap-
plications/quantumyieldstrad.pdf. Quinine bisulfate in 0.5 M H2SO4
was used as a reference for comparison (Φf ) 0.546).40
Indirect Immunofluorescence Detection of TTR. HeLa cells
were plated on coverslips in DMEM containing 10% FBS at 37
°C under 5% CO2 the day before they were transfected with
pcDNA3.1(+)-empty vector or pcDNA3.1(+)-WT-TTR using
Fugene 6 (Roche) according to the manufacturer’s instructions.
After 48 h, the cells were washed with PBS twice before adding
fresh DMEM (without FBS) with A2 (final concentration 10 µM),
and the cells were incubated at 37 °C for 1 h. After two washes
with PBS, the cells were fixed at 25 °C for 15 min with 3.7% (w/
v) paraformaldehyde in PBS. After two washes with PBS, the cells
were permeabilized with 0.2% saponin (w/v) in PBS at 25 °C for
15 min, followed by blocking at 25 °C for 30 min with blocking
buffer (10% goat serum (v/v), 0.2% saponin (w/v) in PBS). Cells
were incubated for 1 h with anti-TTR (a kind gift from Dr. E.
Masliah at UCSD, diluted 1:100) and anti-calnexin (Stressgen,
diluted 1:200), washed three times with PBS, and incubated for
1 h with Alexa Fluor 546 goat anti-rabbit IgG and Alexa Fluor
488 goat anti-mouse IgG (Molecular Probes, A11035 and A11029,
Experimental Section
Fluorimetric Assay with Recombinant Wild-Type Tran-
sthyretin. WT-TTR was expressed in an Escherichia coli expres-
sion system and purified as described previously.39 The covalent
TTR modifier (A1 or A2) or the non-covalent TTR ligand B1 (5
µL of a 1.44 mM solution in DMSO, final concentration 7.2 µM)
was added to 1 mL of a solution of WT-TTR homotetramer (0.2
mg/mL, 3.6 µM) in 10 mM phosphate, 100 mM KCl, and 1 mM
EDTA (pH 7.0 phosphate buffer) in a 2 mL Eppendorf tube. The
samples were vortexed and incubated for 18 h at 25 °C. The
fluorescence changes were monitored using a Varian Cary 50
spectrofluorometer at 20 °C in a 1 cm path length quartz cell. The
excitation slit was set at 5 nm, and the emission slit was set at 5
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respectively) for WT-TTR and calnexin staining. After mounting,
microscopic images were acquired using a fully tunable filter-based
emission collection system (Bio-Rad (Zeiss) Radiance 2100 Rain-
bow laser scanning confocal microscope) that enables the precise
collection of 420-460 nm for compound detection (from a 405
nm laser line). Image analyses were performed using either Zen
2008 Light Edition software (Carl Zeiss MicroImaging) or ImageJ
software (NIH).
Plasmid Constructs Harboring TTR. The pcDNA3.1(+)-WT-
TTR plasmid was produced by ligating the WT-TTR cDNA
(excised from the pCMV5-WT-TTR plasmid36 using HindIII and
XhoI restriction sites) into the pcDNA3.1(+) plasmid (Invitrogen).
A point mutation in the WT-TTR at position 133 (A > G) was
corrected using Quikchange II site-directed mutagenesis (Stratagene)
using the following primers (5′ to 3′): CCGAGGCAGTCCTGC-
CATCAATGTGGC and GCCACATTGATGGCAGGACTGC-
CTCGG. The proper construction of all plasmids was confirmed
by DNA sequencing. Restriction endonucleases and Pfu Turbo DNA
polymerase were purchased from New England Biolabs and
Stratagene, respectively. TTR plasmid DNA was isolated with a
QIAprep Spin Miniprep kit (Qiagen Inc.).
Labeling of Endogenous TTR in Cells. Huh-7 cells or
Gaucher’s patient-derived fibroblasts (Coriell Cell Repositories,
GM08760) were maintained in DMEM containing 10% FBS at 37
°C under 5% CO2. The cells were plated on coverslips in DMEM
(without FBS) the day before compound treatment and imaging.
Cells were washed twice with PBS and then treated with A2 (final
concentration 10 µM) for 30 min at 37 °C in a CO2 incubator.
After two washes, cells were fixed at 25 °C for 15 min with 3.7%
paraformaldehyde (w/v) in PBS, and microscopic images were
acquired as described above.
Monitoring Secretion of WT-TTR from HeLa Cells Utiliz-
ing a Pulse-Chase Experiment. HeLa cells were transfected with
pcDNA3.1(+)-WT-TTR using Fugene 6 (Roche). After 48 h post-
transfection, the cells were washed with PBS twice before adding
fresh DMEM (without FBS) with A2 (final concentration 20 µM).
The cells were incubated at 37 °C for 30 min (pulse) to label the
synthesized WT-TTR, followed by the chase using 10 µM C1 over
a time course of 2 h. After two washes, cells were fixed at 25 °C
for 15 min with 3.7% paraformaldehyde (w/v) in PBS at the
indicated times, and microscopic images were acquired as described
above. The cell culture medium was collected at the indicated time
points during the pulse-chase experiment, and the proteins were
precipitated using a methanol/chloroform mixture (4:1 v/v). The
protein pellet was solubilized in 8 M urea (pH 8), and half of the
mixture was analyzed for the presence of TTR in the medium using
Western blot analysis.
A1 or A2 with the K15A-TTR homotetramer followed by HPLC
analysis revealed no conjugation, consistent with the K15 TTR
residue being essential for chemoselective conjugation (Figure
S1A,B, right panels). The binding of the covalent modifier A2
to K15A-TTR (in the absence of amide bond conjugation) was
confirmed by incubating A2 with K15A-TTR in phosphate
buffer (pH 7), followed by K15A immunoisolation using a
Sepharose resin-conjugated anti-TTR polyclonal antibody.42
After the dissociation of K15A-TTR and bound A2 at high pH,
HPLC analysis showed that 0.88 equiv out of a maximum 2
equiv of A2 was non-covalently bound to K15A-TTR (Sup-
porting Information, Figure S2), an underestimation of bound
A2 owing to the washing steps preceding dissociation.
Furthermore, compound A2 was evaluated to determine its
ability to bind selectively to and then react chemoselectively
with TTR over the 4000+ other proteins in human blood plasma.
Compound A2 (10.8 µM) was incubated with human blood
plasma (TTR concentration is 3.6-5.4 µM) for 24 h at 37 °C
before immunoisolating human TTR using a Sepharose resin-
conjugated anti-TTR polyclonal antibody.42 After dissociation
of TTR from the resin-linked antibody at high pH, HPLC
analysis revealed the ratio of TTR monomer and TTR monomer
covalently attached to the benzoyl portion of A2 via amide bond
formation is effectively 1:1 (taking into account the differences
in molar absorptivity) (Supporting Information, Figure S3A).
Compound A2 must exhibit very high binding selectivity to TTR
in human plasma in order to chemoselectively label 48% of
the TTR subunits through amide bond formation, as only two
of the four subunits comprising the tetramer can be modified
(Figure 1C), corresponding to maximal subunit labeling of 50%.
Mass spectrometric analysis of the conjugate showed the
expected masses (Figure S3B).32 It is clear that these second
generation auxochromic stilbenes exhibit exceptional binding
selectivity and amide-bond-forming chemoselectivity to TTR,
comparable to the first generation stilbenes. This will be
important for determination of plasma TTR concentration and
kinetic stabilizer binding stoichiometry to TTR (see Discus-
sion).32
While incubation of recombinant WT-TTR with A1 or A2
in phosphate buffer (pH 7) for 18 h produced bright blue
fluorescence resulting from amide bond conjugation (Supporting
Information, Figures S4, and Figure 2A, respectively, blue
traces), barely detectable fluorescence resulted from A1 or A2
alone in buffer (Figures S4 and 2A, respectively, red traces).
Notably, no significant fluorescence increase was observed when
A2 was incubated with the recombinant K15A-TTR homotet-
ramer, which binds A2 (Figure S2) but cannot amide bond
conjugate (Figure 2A, black trace). A red shift in the weak but
detectable fluorescence of A1 was observed with K15A-TTR,
providing evidence that it is binding to the T4 binding sites
within K15A-TTR (Figure S4, black trace) but not reacting
(Figure S1A, right panel).
Incubation of unreactive B1 (7.2 µM; Figure 1B), comprising
a built-in amide bond, with WT-TTR (3.6 µM) for 10 min
reveals a significant increase in fluorescence intensity (Figure
2B, blue trace, cf. B1 alone in buffer, red trace). Collectively,
these data support the hypothesis that amide bond linkage of
the benzoyl portion of A1 or A2 to TTR enhances WT-TTR-
(stilbene)2 conjugate fluorescence. In stark contrast to A2, which
remains non-fluorescent upon binding to K15A-TTR, B1
Results
Chemoselective Conjugation of A2 to WT-TTR in Buffer,
Creating Blue Fluorescence. The ability of compounds A1 and
A2 (Figure 1B), auxochromic analogues of previously reported
stilbenes,24,27,30-32,41 to form a chemoselective amide bond with
the K15 residue of TTR was assessed by reverse-phase HPLC
and liquid chromatography-mass spectrometry analysis. Incu-
bating WT-TTR (3.6 µM) with A1 or A2 (7.2 µM, due to the
two thyroxine binding sites per TTR homotetramer) in phosphate
buffer (pH 7) exhibits two peaks of nearly equal intensity
(Supporting Information, Figure S1A,B, left panels). Since only
two of the four subunits comprising the tetramer can be modified
by A2 (Figure 1C), a ∼1:1 peak ratio is the expected result for
binding and chemoselective conjugation (the change in molar
absorptivity of the conjugate was accounted for; note the slightly
increased intensity of the conjugate). Analogous incubation of
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A R T I C L E S
Figure 2. (A) Fluorescence observed after an 18 h incubation of the reactive
stilbene-based TTR modifier A2 with WT- and K15A-TTR. (B) Fluores-
cence observed after a 10 min incubation of nonreactive stilbene B1 with
WT- and K15A-TTR (λex ) 328 nm).
Figure 3. Structures and energetics associated with stilbene photochemistry
and photophysics. (A) Perpendicular singlet excited state (1P*) conformation
required for the trans-to-cis photoisomerization of stilbenes. (B) Relative
energies of the ground (S0) and excited singlet states (S1) of stilbenes as a
function of their conformation (c, cis isoform; t, trans isoform) in solution
(black S1 curve) or when the stilbenes are bound to TTR (red S1 curve),
wherein the perpendicular singlet excited state (1P*) and the cis singlet
excited state (S1) are strongly destabilized by the constraints imposed by
the TTR binding site.
exhibited a ∼4-fold increase in fluorescence intensity upon
binding to K15A-TTR (Figure 2B, black trace), providing
additional evidence that this family of stilbenes binds to K15A-
TTR. The fluorescence intensity was diminished relative to WT-
TTR binding, presumably due to changes in the T4 binding site
environment or dynamics associated with the K15A mutant.43
We also incubated A2 with recombinant WT- or K15A-TTR,
and the resulting solution fluorescence was detected using a
hand-held UV lamp (365 nm). While intense blue fluorescence
resulted from the incubation of WT-TTR with A2 (Supporting
Information, Figure S5, vial 1), no fluorescence was observed
when K15A-TTR was incubated with A2 (vial 2). In contrast,
intense blue fluorescence was produced when amide B1 was
incubated with either WT-TTR or K15A-TTR (Figure S5, cf.
vials 3 and 4, respectively). The reaction chemoselectivity of
A2 for WT-TTR was further demonstrated by the observation
that WT-TTR, but not K15A-TTR, was detected by conjugate
fluorescence on an SDS-PAGE gel (Supporting Information,
Figure S6).
we hypothesize that stilbene A2 could be especially useful for
cellular imaging. What makes A2 atypical in this regard? To answer
this question, it is useful to consider the excited-state relaxation
mechanism available to most stilbenes.44-49
Upon promotion of an electron from the stilbene HOMO to
the LUMO, a singlet excited state is formed that repopulates
the ground state in solution primarily by isomerization to a cis-
stilbene, and to a lesser extent by stilbene fluorescence.44-49
The trans form of the singlet excited state can twist about the central
C-C bond orienting the planes of the two aryl rings perpendicular
to each other (Figure 3A), affording a mixture of cis and trans
isomers after internal conversion and conformational relaxation.44-49
While TTR can bind to small molecules composed of two aromatic
Investigating the Mechanism by Which Stilbene Conjuga-
tion to TTR Creates Blue Fluorescence. The data outlined above
and presented below suggest that amide bond conjugation is
required for the observation of TTR-(stilbene)ne2 conjugate fluo-
rescence. As mentioned above, it is unusual that stilbenes remain
dark when bound to proteins, including TTR, and in this regard
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Choi et al.
rings when the tethered rings are up to 40° out of plane (based on
TTR-stilbene crystal structures24,27,30-32,50), it would be energeti-
cally unfavorable to bind the excited singlet state in a conforma-
tion where the two aryl rings are oriented perpendicular to one
another. Thus, we propose that the increase in fluorescence
quantum yield of B1 from 0.09 in buffer to 0.55 in complex
with TTR results from the resculpted excited singlet-state energy
surface of the stilbene in complex with TTR, such that the trans
conformer is the only state of relatively low potential energy
(Figure 3B),44-49 preventing photoisomerization-based relax-
ation of B1. That B1 binding to TTR prevents B1 photoisomer-
ization is consistent with the observation that the irradiation of
B1 in buffer (Life Technologies, TFX-35M, 312 nm) for 10 s
affords 60% of the cis isomer, along with other minor photo-
reaction products (Supporting Information, Figure S7A), whereas
irradiation of B1 bound to TTR yields only 20% cis isomer
(Figure S7B).
It appears that the increase in fluorescence quantum yield of
A2 from 0.00 in buffer to 0.27 in the TTR-(stilbene)ne2
conjugate is only partly explained by the mechanism proposed
for B1.44-49 That A2 is different from B1 is supported by
observation that A2 does not readily form the cis isomer or
undergo photoreactions upon irradiation in buffer for 10 s
(Figure S7C). Nor does A2 fluoresce upon binding to K15A-
TTR (Figure 2A). It is also notable that the fluorescence intensity
of A2 does not increase appreciably in dichloromethane
(Supporting Information, Figure S8A), unlike B1, which is an
environmentally sensitive fluorophore (Figure S8B). The very
low fluorescence intensity of A2 combined with its resistance
to photoisomerization suggests that the thioester comprising A2
quenches its fluorescence, both in solution and when bound to
K15A-TTR. However, upon amide bond formation with TTR,
the conjugate derived from A2 becomes fluorescent because the
stilbene is no longer quenched by the thioester functionality
and because the perpendicular excited singlet-state conformation
cannot be accommodated in the TTR-(stilbene)ne2 conjugate
structure (Figure 3B), preventing the photoisomerization-based
relaxation. The formation of the perpendicular excited singlet-
state conformation required for photoisomerization (Figure 3A)
is envisioned to be even more energetically inaccessible in the
amide bond conjugate, as the aryl ring occupying the inner T4
binding subsite is rigidly held through complementary non-
covalent interactions24,27,30-32,41,50-63 and the ring occupying
the outer T4 binding subsite is covalently tethered to the protein
through a m-amide linkage.32 The thioester stilbene quenching
hypothesis is supported by observations that A1 and B1 are
quenched, dose dependently, by the addition of millimolar
concentrations of thiophenol (Supporting Information, Figure
S9).
Antibodies that bind to stilbenes that have aromatic side chains
in their stilbene binding sites can form either fluorescence exci-
plexes or charge-transfer-based luminescent antibody-stilbene
complexes.64,65 However, the TTR stilbene binding sites do not
comprise any aromatic residues, making an exciplex fluorescence
or charge-transfer luminescence explanation unlikely.64,65
Summing up the mechanistic details, A2 remains dark, even
if bound to proteins, including TTR, because of a thioester
quenching mechanism. Upon amide bond conjugation to TTR,
the fluorescence quantum yield of the blue conjugate is
reasonably high because the trans-to-cis photoisomerization
mechanism is energetically disfavored owing to TTR binding
and photobleaching is minimized by the lower energy excitation
and emission resulting from the dimethylamino auxochromic
substituent on one of the aromatic rings.
Kinetics of TTR Fluorescent Conjugate Formation at 37
°C in Human Cell Lysate. Since the one-chain, one-binding-
site TTR tag being created is envisioned to be useful for doing
pulse-chase experiments in the secretory pathway of eukaryotic
cells, the kinetics of WT-TTR modification by A2 in concen-
trated cell lysate was investigated (Figure 4A). HeLa cell lysate
was employed because these cells do not make transthyretin.
We added WT-TTR at a final concentration of 2 µM (tetramer)
to the HeLa cell lysate (2 µg/µL total protein concentration) to
which A2 (6 µM) was added. At 37 °C, the conjugate formed
with a t50 of 18 min, within experimental error of the t50 (19
min) of TTR (2 µM) conjugate formation with A2 (6 µM) in
phosphate buffer (pH 7) (Figure 4A). That A2 remains dark,
even if bound to other proteins in the absence of TTR, is
demonstrated by the HeLa cell lysate (2 µg/µL total protein
concentration) incubated with A2 (2 µM) for 180 min (Figure
4A, red trace).
Selectivity of the A2 TTR Conjugation Reaction at 37 °C
in Human Cell Lysate. The ability of A2 to form a chemose-
lective amide bond with the K15 residue of TTR in cell lysate
was assessed by RP-HPLC and LC-MS analysis. Incubating
WT-TTR (5 µM) with A2 (15 µM) in 900 µL of HeLa cell
lysate (2 µg/µL total protein concentration, excluding TTR)
exhibits peaks of nearly equal intensity (Figure 4B; the molar
absorptivity changes associated with benzoylation were ac-
counted for), demonstrating high binding selectivity and a highly
chemoselective amide bond forming reaction with TTR (49%
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A Stilbene Fluorescent Tag for Transthyretin
A R T I C L E S
TTR, and not cells transfected with empty vector (control),
afforded strong conjugate fluorescence upon A2 treatment
(Figure 5A, first column). Indirect TTR immunofluorescence
(Figure 5A, second column) afforded an analogous image,
revealing that TTR was being imaged by A2 treatment. TTR
conjugate fluorescence co-localized with calnexin indirect
immunofluorescence, a marker for the endoplasmic reticulum
(ER), confirming that WT-TTR within the secretory pathway
is being observed (Figure 5A, right most column, co-localization
artificially colored white). TTR conjugate fluorescence is also
expected in the Golgi and in secretory vesicles, explaining why
TTR conjugate fluorescence is observed in organelles in addition
to the ER.
We also demonstrated that endogenous TTR could be
detected with A2-derived fluorescence in a common cell line
secreting TTR, which is challenging, as the low quantities
of TTR being detected are undergoing folding and tetramer-
ization in the endoplasmic reticulum, vesicular trafficking
to the Golgi, followed by vesicular trafficking to the plasma
membrane and release into the media.36 While A2 treatment
gave no significant fluorescence in human fibroblasts, con-
sistent with reports that these cells do not make TTR (Figure
5B, bottom panels, and Figure S10B),66 the A2-treated Huh-7
human liver (hepatoma) cells afforded strong fluorescence
that co-localized with calnexin immunofluorescence (Figure
5B, top panels). The confocal fluorescence observations are
consistent with substantial evidence that the liver secretes
the vast majority, if not all, of the TTR into the human
bloodstream.67 That TTR could be visualized in the secretory
pathway addresses the sensitivity of A2-derived TTR fluo-
rescent conjugate-based detection.36
Pulse-Chase Experiment Demonstrating WT-TTR Secre-
tion from HeLa Cells. Since imaging WT-TTR in the secretory
pathway of HeLa cells post-transfection employing A2 is
feasible (Figure 5A) and A2 conjugation kinetics in HeLa cell
lysate (Figure 4A) is sufficiently rapid, we next utilized A2-
based fluorescence to label WT-TTR in a pulse-chase experi-
ment in living, transiently transfected HeLa cells. WT-TTR in
HeLa cells (48 h post-transfection) was labeled using a 20 µM
A2 pulse for 30 min, followed by a chase employing C1 (10
µM) over a 2 h time course (Figure 6). C1 is a non-fluorescent,
highly selective, non-covalent, dihydrostilbene-based TTR
binder31 (see Figure 6A for a line drawing of its structure). While
notable TTR conjugate fluorescence was observed 30 and 60
min into the chase period, no conjugate fluorescence was
detected after 120 min (Figure 6C, first column), indicating that
the WT-TTR has been secreted out of the cells over that period
(Figure 6B). The movement of TTR through the secretory
pathway in our pulse-chase experiment (Figure 6C) is consis-
tent with previous data from BHK and MMH cells transiently
overexpressing WT-TTR, demonstrating that WT-TTR secretion
takes about 90 min.36 It appears that the 20 min t50 for TTR
fluorescent conjugate formation observed in HeLa cell lysate
(Figure 4A) is a reasonable facsimile for what happens in a
transiently transfected living HeLa cell, as a 30 min pulse
afforded a strong signal in the pulse-chase experiment (Figure
6C) that disappeared over time as a consequence of the C1 chase
and secretion on the expected 2 h scale, with TTR secretion
Figure 4. Kinetics and chemoselectivity of the A2 conjugation reaction
with WT-TTR in HeLa cell lysate vs buffer. (A) Time dependence of
fluorescent conjugate formation, created by reacting A2 and WT-TTR in
phosphate-buffered saline (pH 7; green trace). Time-dependent fluorescence
changes or lack thereof occurring when HeLa cell lysate (lacking TTR) is
incubated with A2 alone (red trace) or when HeLa cell lysate is incubated
alone with added TTR (without A2, blue trace) or when HeLa cell lysate
with added WT-TTR is treated with A2 at 37 °C (black trace). (B) RP-
HPLC analysis of TTR immunoisolated from HeLa cell lysate utilizing an
anti-TTR antibody conjugated to resin. The nearly equal peak areas resulting
from the unmodified subunits and the conjugated subunits (the molar
absorptivity changes associated with benzoylation were accounted for)
demonstrate high binding selectivity and a highly chemoselective amide
bond-forming reaction with TTR (49% out of a maximum of 50% of the
subunits were modified).
out of a maximum of 50% of the subunits were modified, Figure
1C). Immunoisolation of TTR affords the expected mass
resulting from conjugate formation (14 185 m/z [M + H]+ for
benzoylation by A2). The equal rates of TTR conjugate
formation in HeLa cell lysate and in buffer (Figure 4A,B)
demonstrate that the binding selectivity of A2 to TTR and the
chemoselectivity of amide bond conjugation to TTR are
excellent in complicated biological environments.
Detection of WT-TTR in Transfected HeLa Cells and in
Huh-7 Human Liver Cells. Having demonstrated that A2 is a
non-fluorescent stilbene in solution (Figure 2A) and in HeLa
cell lysate not containing TTR (Figure 4A, red trace), we next
investigated whether specific labeling of TTR could be achieved
in the complex environment of the eukaryotic cellular secretory
pathway. Secreted WT-TTR was produced in HeLa cells by
transient transfection, and the cells were treated with A2 for
1 h prior to imaging using confocal fluorescence microscopy.
The expression of WT-TTR in the HeLa cells after transient
transfection was confirmed using Western blot analysis (Sup-
porting Information, Figure S10A), as was the absence of TTR
in the empty vector control. Only HeLa cells expressing WT-
(66) Su, A. I.; Wiltshire, T.; Batalov, S.; Lapp, H.; Ching, K. A.; Block,
D.; Zhang, J.; Soden, R.; Hayakawa, M.; Kreiman, G.; Cooke, M. P.;
Walker, J. R.; Hogenesch, J. B. Proc. Natl. Acad. Sci. U.S.A. 2004,
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Choi et al.
Figure 5. (A) Confocal fluorescence microscopy images of A2 (10 µM)-treated HeLa cells transfected with empty vector (top panels) or WT-TTR (bottom
panels). (B) Confocal fluorescence miscroscopy images of Huh-7 cells (liver) (top panels) and fibroblasts (bottom panels) 30 min after 10 µM A2 treatment.
In (A) and (B), the fluorescence from A2 is artificially colored red, WT-TTR was detected by indirect immunofluorescence (green staining), and the endoplasmic
reticulum was labeled using indirect calnexin immunofluorescence (artificially colored gray). The extreme right column shows the colocalization between
the A2-derived fluorescence and calnexin (artificially colored white).
into the media being confirmed (Figure 6B). The reaction
kinetics of A2 would become problematic if time resolution
higher than tens of minutes were required for a particular
application.
The TTR concentration in plasma is an established metric of
nutritional status and is an often used clinical laboratory test.68
A2 demonstrates sufficient binding selectivity to TTR in human
plasma and amide bond reaction chemoselectivity with Lys-15
in TTR (Figure S3A,B) to create a new method that would
replace the error-prone immunoprecipitation-turbidity assay
widely used in clinical laboratories today to quantify TTR
plasma levels.
Another immediate application of A2 is the quantification
of kinetic stabilizer binding stoichiometry to rare variants
of TTR in human plasma.42 Tafamidis, a TTR kinetic
stabilizer that we discovered,52 has recently been shown in
a phase II/III placebo-controlled clinical trial to halt the
progression of familial amyloid polyneuropathy (FAP).
Negatively cooperative binding of Tafamidis to one of the
two T4 binding sites in the TTR tetramer is sufficient to
increase the kinetic barrier for tetramer dissociation (through
selective ground state stabilization) such that it becomes
insurmountable under physiological conditions, preventing
amyloidogenesis that causes FAP.29,69-71 While the human
oral dose required for occupancy of more than one TTR
binding site is known for the most common TTR FAP-
associated mutations, this has not been established for all
mutations. It is now feasible to utilize A2-derived conjugate
fluorescence to measure the stoichiometry of kinetic stabiliz-
ers, such as Tafamidis, bound to TTR in patient plasma using
Discussion
We describe a non-fluorescent, second generation, auxochro-
mic stilbene A2 that very selectively binds to transthyretin in
the eukaryotic cellular secretory pathway (remains dark) and
then chemoselectively reacts with a pKa-perturbed Lys-15
ε-amino group in TTR, creating a bright blue fluorescent
conjugate. Stilbene A2 exhibits sufficiently rapid TTR conjuga-
tion kinetics at 37 °C to enable pulse-chase experiments to be
performed within the secretory pathway of HeLa cells, dem-
onstrating that transthyretin can be imaged as it transits through
the secretory pathway. The progress reported is viewed as a
first and necessary step toward our long-term goal of creating
a one-chain, one-binding-site transthyretin tag that can be used
in the eukaryotic secretory pathway to follow protein secretion
and turnover utilizing A2 or an analogous molecule. While the
reaction kinetics of A2 with TTR are well-suited for studying
the folding, trafficking, secretion, and degradation of TTR and
TTR-tagged proteins, other thiol-containing functional groups
(required to keep the latent fluorophores dark) with faster
reaction kinetics are being explored to carry out pulse-chase
experiments with higher time resolution.
An immediate application of A2 is to utilize the conjugate
fluorescence to quantify TTR concentration in human plasma.
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A Stilbene Fluorescent Tag for Transthyretin
A R T I C L E S
Figure 6. Secretion time course of WT-TTR from HeLa cells monitored utilizing a pulse-chase experiment. HeLa cells were transfected with WT-TTR
and after 48 h were treated with 20 µM A2 for a 30 min pulse. The secretion time course of labeled WT-TTR was followed by chasing the cells with the
selective, noncovalent, nonfluorescent TTR kinetic stabilizer C131 (10 µM) over a chase period of 2 h. (A) Structure of the chase compound, C1. (B)
Western blot demonstration that WT-TTR was secreted into the cell culture media over the same time course that TTR left the secretory pathway. (C)
Confocal fluorescence microscopy images wherein the fluorescence from A2 is artificially colored red (column 1). The expression of WT-TTR in the HeLa
cells only after transfection was confirmed by indirect immunofluorescence (green staining, column 2) as a function of time, and the endoplasmic reticulum
was labeled using indirect calnexin immunofluorescence (artificially colored gray, column 3). The extreme right column shows the co-localization between
the A2-derived TTR conjugate fluorescence and calnexin (artificially colored white) as a function of time.
Acknowledgment. We thank the NIH (DK46335) as well as
the Skaggs Institute for Chemical Biology and the Lita Annen-
berg Hazen Foundation for financial support and Prof. David
Millar at Scripps, Prof. Alice Ting at MIT, and the reviewers
for helpful comments. Technical support from Dr. Tingwei Mu,
Dr. Steve Bourgault, and Mike Saure is also greatly appreciated.
an A2-kinetic stabilizer competition assay. After assay
optimization, physicians could use such an approach to titrate
the dose of kinetic stabilizer to be used in patients with rare
mutations to be sure that the kinetic stabilizer occupies at
least one TTR binding site per tetramer.57 Since there are
over 100 TTR mutations that cause FAP, developing the A2
method for determining kinetic stabilizer TTR binding
stoichiometry is important and especially timely, since
Tafamidis, the first kinetic stabilizer to be shown to be
efficacious, is expected to become a regulatory agency
approved drug during the coming year.52,57,69,70
Supporting Information Available: Procedures for the
reverse-phase HPLC analyses; binding stoichiometry mea-
surement; photoisomerization; detection of TTR labeling by
SDS-PAGE; Western blot analyses; synthesis of A1, A2,
and B1; and additional data referred to in the text. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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