Minimalist probes for studying protein dynamics: Thioamide quenching of selectively excitable fluorescent amino acids

Article abstract of DOI:10.1021/ja3005094

Fluorescent probe pairs that can be selectively excited in the presence of Trp and Tyr are of great utility in studying conformational changes in proteins. However, the size of these probe pairs can restrict their incorporation to small portions of a prot

Full text of DOI:10.1021/ja3005094

Communication
pubs.acs.org/JACS
Minimalist Probes for Studying Protein Dynamics: Thioamide
Quenching of Selectively Excitable Fluorescent Amino Acids
Jacob M. Goldberg, Lee C. Speight, Mark W. Fegley,† and E. James Petersson*
Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United States
S
* Supporting Information
therefore limited to use in model proteins under highly purified
ABSTRACT: Fluorescent probe pairs that can be
selectively excited in the presence of Trp and Tyr are of
great utility in studying conformational changes in
proteins. However, the size of these probe pairs can
restrict their incorporation to small portions of a protein
sequence where their effects on secondary and tertiary
structure can be tolerated. Our findings show that a
thioamide bonda single atom substitution of the peptide
backbonecan quench fluorophores that are red-shifted
from intrinsic protein fluorescence, such as acridone. Using
steady-state and fluorescence lifetime measurements, we
further demonstrate that this quenching occurs through a
dynamic electron-transfer mechanism. In a proof-of-
principle experiment, we apply this technique to monitor
unfolding in a model peptide system, the villin headpiece
HP35 fragment. Thioamide analogues of the natural amino
acids can be placed in a variety of locations in a protein
sequence, allowing one to make a large number of
measurements to model protein folding.
conditions. However, the observation that Trp is quenched by
thioamides without appreciable spectral change suggested the
possibility of finding a fluorophore that could be selectively
excited in a protein and could also be quenched by a thioamide.
Mechanistically, such a process might involve a photoinduced
electron transfer (PET) mechanism, rather than a Forster
̈
resonance energy transfer (FRET) or Dexter transfer
mechanism, since FRET and Dexter require spectral overlap
between the donor and acceptor chromophores.5 [Note:
Thioamide residues are represented by the one- or three-letter
codes of the equivalent oxoamide amino acids with a prime
symbol (e.g., A′ or Ala′ represents thioalanine).]
We sought a robust fluorophore with a large extinction
coefficient and a high quantum yield that would be compact
enough to be tolerated in several positions in a protein
sequence and could be incorporated genetically. We began by
studying three fluorescent scaffolds that met these general
requirements: 7-azaindole, 7-methoxycoumarin, and acridone,
and their amino acid derivatives 7-azatryptophan (7Aw or ω),
7-methoxycoumarin-4-ylalanine (Mcm or μ), and acridon-2-
ylalanine (Acd or δ), respectively (Figure 1). In addition to
their favorable optical properties, each of these amino acid
scaffolds has been shown to be compatible with in vivo or in
vitro ribosomal incorporation and has previously been used to
probe biomolecular structure.6 We expect them to be minimally
perturbing as compared to other fluorophores since 7Aw is
isosteric with Trp, Mcm is only 29% larger, and Acd is 47%
larger than Trp; fluorescein is more than twice the size of Trp,
and Texas Red is more than three times larger.
Initially, we examined the fluorescence quenching of each
chromophore by aqueous thioacetamide. We prepared dilute
(<50 μM) solutions of each fluorophore in 50 mM
thioacetamide in phosphate buffer (100 mM, pH 7.00) and
measured their fluorescence. As a control, we measured the
fluorescence of equimolar solutions of the fluorophores in the
absence of quencher (in phosphate buffer only) and in the
presence of 50 mM acetamide. In all cases, we observed
substantial quenching upon addition of thioacetamide and no
quenching upon addition of acetamide. To quantify the extent
of quenching, we calculated the quenching efficiency, EQ(TA),
at the wavelength of maximum emission (Figure 1; see
Supporting Information for calculations). Since concentrated
thioacetamide solutions absorb light very strongly below 350
nm, the observed quenching efficiencies of 7Aw and Mcm may
luorescence spectroscopy is a powerful tool for studying
F
dynamic processes in biological systems.1 To harness the
full potential of this technique, red-shifted fluorophores are
often required that can be excited independently of the intrinsic
fluorescence background of native proteins.2 When these
experiments are used to monitor a distance-dependent event,
such as a conformational rearrangement, a second probe is
typically required that can act as an energy donor or acceptor.3
Because of their bulk, introducing such probe pairs at arbitrary
positions in a protein sequence risks significantly perturbing
native structure and function. If one of the partners in the
probe pair were sufficiently nonperturbing to be tolerated at
almost any position in a protein, much more detailed
information could be obtained. Here, we show that a thioamide,
a single-atom substitution of the peptide backbone, can serve as
such a partner for the red-shifted fluorophores azaindole,
coumarin, and acridone. Furthermore, we demonstrate that the
acridone is quenched through dynamic electron transfer and
the acridone/thioamide probe pair can be used to monitor
conformational changes in proteins.
Previously, we have shown that thioamides quench intrinsic
protein fluorescence arising from excitation of tryptophan (Trp
or W) or tyrosine (Tyr or Y), as well as extrinsic protein
fluorescence arising from excitation of p-cyanophenylalanine
(Cnf or F*).4 These fluorophores cannot be selectively excited
in proteins containing other Trp or Tyr residues and are
Received: January 16, 2012
Published: April 3, 2012
© 2012 American Chemical Society
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Figure 2. Stern−Volmer quenching of acridon-2-ylalanine (Acd) by
thioacetamide. (Left) Fluorescence lifetime measurements of 2 μM
Acd in 100 mM phosphate buffer, pH 7.00 at 25 °C in the presence of
Figure 1. Chromophore structures and spectral properties. Chemical
structures of 7-azatryptophan (7Aw), 7-methoxycoumarin-4-ylalanine
(Mcm), acridon-2-ylalanine (Acd), and a thioamide analogue of
alanine (A′ or Ala′) with corresponding wavelengths of maximum
absorbance (λmax) and emission (λem). Wavelengths for selective
excitation in a protein are listed as λex. Thioamide quenching
efficiencies listed as EQ(TA) are for dilute solutions of the fluorophore
in 50 mM thioacetamide, and EQ(P2) or as EQ(G2) are those in a short
thioamide containing diproline or diglycine peptide, respectively.
○
50 mM acetamide (×, blue fit) or 50 mM thioacetamide ( , red fit).
Heavy lines are single exponential fits to the raw data. (Right) Stern−
Volmer plots of Acd fluorescence intensity (green circles) and lifetime
(black triangles) as a function of thioacetamide concentration; error
bars represent standard error. Linear fits to the Stern−Volmer
equation are shown (see Supporting Information).
We calculated the quenching constant as kQ = 2.6 × 109 M−1
s−1, near the value predicted for a diffusion-limited, bimolecular
quenching rate constant predicted by the Smoluchowski
equation: k2 = 8.3 × 109 M−1 s−1.11 Although it is possible to
assign the discrepancy in the values of kQ and k2 to a quenching
efficiency factor f Q = 0.31, it is also possible that the theoretical
model does not describe the system perfectly. We examined the
UV/vis absorbance spectra of Acd solutions in the presence and
absence of concentrated thioacetamide. We observed no
difference in the absorbance of these samples, suggesting that
no static, ground-state complex is formed with thioacetamide
(see Supporting Information). These data point to an
exclusively dynamic PET quenching mechanism.
be overestimates. Nonetheless, these results indicated that
thioamides can quench each fluorophore.
To ensure that these results would translate to a protein
system, we synthesized several peptides bearing a fluorophore
at the C-terminus and either leucine or thioleucine at the N-
terminus, separated by two proline residues or two glycine
residues. Again, we calculated quenching efficiencies, EQ(P2)
and EQ(G2), for each flourophore by comparing the
fluorescence of the oxo- and thioamide-containing peptides
(Figure 1). For 7Aw, EQ(G2) was much smaller than EQ(50
mM), likely due to the absence of the inner filter effect in the
peptide system. In all cases, much greater quenching was
observed in the glycine peptides than in the proline peptides.
This is consistent with a collisional quenching mechanism that
is enabled by a more flexible linker between fluorophore and
thioamide.7 For further experiments, we chose to focus on the
quenching of Acd rather than 7Aw or Mcm because of its high
quantum yield (ΦAcd = 0.95;8 Φ7Aw = 0.01;9 ΦMcm = 0.1810).
We measured the emission of Acd in varying concentrations
of thioacetamide using variable-temperature fluorometry at 10,
25, 35, 45, and 65 °C. At each temperature, the fluorescence
intensity varied inversely with thioacetamide concentration and
appeared to follow Stern−Volmer quenching behavior (Figure
2). The Stern−Volmer constant, KSV, icreased with temperature
from 34.16 0.55 M−1 at 10 °C to 53.51 0.40 M−1 at 65 °C,
suggesting that thioamide quenching of Acd may arise from
dynamic electron transfer.
To evaluate the energetic feasibility of a PET mechanism, we
calculated the Gibbs energy for transferring an electron from
thioacetamide to the excited fluorophore (ΔGET).12 We
measured the electrochemical properties of acridone in
anhydrous, deoxygenated DMF using cyclic voltammetry and
observed irreversible oxidation (Epa = 1.4 V vs SCE) and
reduction (Epc = −1.9 V vs SCE) events. Using these values, the
reported oxidation potential of thioacetamide (0.97 V vs
SCE),13 and the lowest energy, vibrational zero−zero electronic
excitation of acridone (E0,0 = 3.0 eV), we calculated ΔGET
=
−0.13 eV according to the Rehm−Weller equation (see
Supporting Information).14 A favorable ΔGET implies that
transfer of an electron from a thioamide to excited Acd is
possible. A similar analysis using previously reported values for
the reduction potential and E0,0 of 7-methoxycoumarin provides
a value of ΔGET = −0.6 eV.15 We were unable to obtain
satisfactory electrochemical data to perform a calculation of
ΔGET for 7Aw.16 It is also possible that PET occurs in the other
direction: the electron may be transferred from the fluorophore
to the thioamide, but our current electrochemical data do not
allow us to calculate ΔGET for this process.
Time-correlated single photon counting (TCSPC) spectros-
copy on freshly prepared Acd solutions at 25 °C was employed
to further characterize the mechanism of quenching. In 100
mM phosphate buffer, pH 7.00, we found the lifetime of Acd to
be 15.73 0.05 ns. The lifetime was unchanged upon addition
of 50 mM acetamide (τ = 15.76 0.05 ns), but decreased to
As a demonstration of the utility of the Acd/thioamide probe
pair, we studied the thermal denaturation of a variant of the
villin headpiece subdomain that has been well characterized,
HP35.17 HP35 adopts a compact folded structure with the N-
and C-termini within 20 Å.18 Previously, we used a FRET
interaction between C-terminal Cnf35 and N-terminal Leu′1 in a
double-labeled version of HP35 to monitor unfolding.4a We
made this HP35-L′1F23F*35 construct without Trp (W23F
5.13
0.02 ns upon addition of 50 mM thioacetamide. At
intervening concentrations of thioacetamide, the lifetime varied
with the concentration of quencher according to Stern−Volmer
kinetics, with a Stern−Volmer constant KSV = 41.36
0.05
M−1, nearly identical to that found from steady-state measure-
ments at 25 °C, KSV = 41.96 0.42 M−1 (Figure 2). In all cases,
lifetime data could be fit to single exponential functions.
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mutation) because spectral overlap of Cnf with Trp makes
deconvoluting the contributions of FRET to Trp and the
thioamide difficult.4b,19 The HP35 construct used in this study,
HP35-L′1δ35 (containing a Trp at position 23), does not suffer
this problem since Acd can be excited at wavelengths that do
not excite Trp. Therefore, we can use PET quenching of Acd
by the thioamide to track unfolding. In the following
experiments, we compare double-labeled HP35-L′1δ35 to
oxopeptide HP35-δ35 using temperature-dependent circular
dichroism (CD), steady state fluorescence, and TCSPC.
CD spectroscopy indicated that both proteins were primarily
α-helical and underwent unfolding events that could be
modeled as cooperative two-state transitions with comparable
Tm values (53 °C for HP35-δ35 and 56 °C for HP35-L′1δ35).
These thermal denaturation curves were used to determine the
fraction of each peptide that was folded at any given
temperature. Steady-state fluorescence measurements on
equimolar solutions of the proteins in phosphate-buffered
saline, pH 7.00, showed that Acd in the thioamide-containing
variant was quenched 16% relative to the all oxo-amide
containing protein at 1 °C. The quenching efficiency (EQ)
increased gradually with temperature, reaching a maximum of
22% at 66 °C, and then decreased rapidly to 15% at 95 °C.
The dependence of EQ on temperature can be displayed with
respect to fraction folded as shown in Figure 3. Several
observations are worth noting from these transformed data,
which are plotted with data from our previous HP35-L′1F23F*35
experiment for comparison. First, there is a sharp initial increase
in quenching efficiency that occurs while the protein retains a
mostly folded state. We attribute this to a slight increase in the
flexibility of the N- and C-termini, favoring contact between the
chromophores that leads to quenching. As the protein begins to
unfold, EQ increases smoothly, presumably as a result of the
increased rates of productive collisions that are accessible at
higher temperatures. EQ drops dramatically when the protein
globally unfolds at fraction folded of 0.2, consistent with the
increase in separation between the ends of the protein seen in
the HP35-L′1F23F*35 FRET experiments. Previous experiments
have shown that repacking of the HP35 core can occur well
before global unfolding, consistent with our observations.20
Specifically, two other groups have recently observed separation
of the villin N- and C-termini under partially denaturing
conditions using triplet−triplet energy transfer.21
Figure 3. Thermal denaturation of HP35. (Top left) Structure of
HP35-Leu′1Acd35 modified from PDB 1VII and rendered in PyMOL
(Schrodinger, Portland, OR). (Top right) Steady-state quenching
̈
efficiency (EQ(Acd), black circles) as a function of fraction of protein
folded determined by CD spectroscopy and the separation of the N-
and C-termini as a function of fraction folded determined previously
by a FRET experiment with thioamide quenching of Cnf
(RFRET(CNF), green squares). (Bottom left) Normalized fluorescence
○
lifetime measurements of Acd in HP35-L′1δ35 ( , red fit) and HP35-
Acd35 (×, blue fit) at 55 °C. Average lifetimes are listed as determined
by fits to a biexponential model as described in the Supporting
Information. (Bottom right) Average lifetimes of Acd in HP35-L′1δ35
(red circles) and HP35-δ35 (blue squares) as a function of fraction
folded and determined from biexponential fits to the data. Quenching
efficiency calculated by comparing lifetime measurements of the oxo-
and thio-proteins as a function of fraction folded (purple triangles).
Error bars are estimated from the fits.
biexponential model, in which 5% quenching was observed in
the thioamide-containing peptide.
Since the data from HP35-δ35 could not be fit to a single
exponential, we measured the fluorescence lifetime of Acd in
other all oxo-amide systems to determine whether this resulted
from incorporation of the amino acid into a peptide backbone.
For both the LP2δ (τ = 16.68 0.05 ns at 25 °C) and LG2δ (τ
Fluorescence lifetime measurements of the oxo and thio
versions of HP35 at several temperatures are generally
consistent with the steady-state observations (Figure 3). The
magnitude of EQ as determined by comparing the average
lifetimes of HP35-δ35 and HP35-L′1δ35 is less than that obtained
by comparing steady-state measurements, but the overall trend
as a function of fraction folded is the same. The change in
magnitude of EQ might indicate that there is a static quenching
component in the protein context that we did not observe in
bimolecular thioacetamide quenching experiments.
= 16.35
0.04 ns at 25 °C) peptides described above, we
found that the Acd lifetime data for the oxo-peptides could be
fit with a simple single exponential function, but a satisfactory
fit to the corresponding data for the thiopeptides could be
achieved with a biexponential model. We observed a
substantially shorter average lifetime for the L′G2δ (τavg
=
Although we were able to fit the lifetime data collected for
the free amino acid in the presence and absence of
thioacetamide to a single exponential, we were not able to
model the lifetime of Acd in HP35 with this function. Instead,
we found that a biexponential fit described the data well, giving
average lifetimes of 15.76 0.25 ns for HP35-δ35 and 15.38
0.24 ns for HP35-L′1δ35 at 25 °C. We used this function as a
simple model of a system that placed Acd in at least two
environments with distinct quenching. Gaussian fits to the data
produced results nearly identical to those found with the
12.92 0.16 ns at 25 °C) than for L′P2δ (τavg = 16.08 0.28 ns
at 25 °C), consistent with steady-state measurements and the
idea that access to favorable conformations is important for
quenching. In a separate experiment, we found that the
presence or absence of C-terminal amidation also does not
seem to have an effect on the oxo-peptide: both LP2δ-OH (τ =
16.68 0.05 ns) and LP2δ-NH2 (τ = 16.67 0.05 ns) could be
fit to a single exponential model.
The difference between the lifetimes of Acd in HP35-δ35 and
LP2δ or LG2δ shows that there is some quenching of Acd in
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HP35 independent of the thioamide. This is likely to be true in
any real protein, where almost every fluorophore experiences
some quenching when located near redox-active amino acids
such as Trp, Tyr, His, Met, and Cys.22 Since the HP35 proteins
studied here differ only by the single O to S substitution in the
Leu′ amide bond, we can assign quenching to be specifically
due to the thioamide. It is crucial that measurements on
thioamide-containing proteins be compared to the all oxo-
amide version to control for the effects of temperature, solvent
accessibility, and neighboring residues on fluorescence that may
arise during a conformational change. With these controls, one
can follow the unfolding of events specific to the thioamide site
with fluorescence spectroscopy. At this point, we do not
attempt to assign interchromophore distances on the basis of
the efficiency of quenching; rather we merely interpret
quenching as resulting from transient molecular contact so
that fluorophores are less than 20 Å apart if quenching is
observed. Further mechanistic study may allow us to assign
distances in a more precise manner.
In conclusion, we have demonstrated that thioamides are
capable of quenching a variety of fluorophores, including some
that can be selectively excited in the context of a protein. Our
findings from steady-state and lifetime measurements of Acd
fluorescence indicate that this quenching arises through a
dynamic photoinduced electron transfer mechanism. Further-
more, we have shown that the Acd-thioamide fluorophore−
quencher probe pair can be used to study conformational
changes in proteins. Although previous experiments have
gained valuable information using visible wavelength fluores-
cent donors and Trp quenchers, we believe that our reporter
system will allow us to study protein dynamics in far greater
detail because the thioamide will be tolerated at more positions
in the protein.12,23 Other recent work in our laboratory has
demonstrated that thioamides can be incorporated into full-
sized proteins through native chemical ligation, so that studies
of the type conducted on HP35 could be carried out on larger
proteins.24 Taken together, these studies lay the groundwork
for the application of thioamide quenching to the study of
protein folding and the design of conformational biosensors.
use of the fluorometer, major instruments are supported by
NSF DMR05-20020 and NSF MRI-0820996. We thank Tom
Troxler for assistance with the TCSPC measurements collected
at the Regional Laser and Biomedical Technology Laboratories
at the University of Pennsylvania (supported through NIH/
NCRR P41RR001348).
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ASSOCIATED CONTENT
* Supporting Information
Descriptions of peptide and small molecule synthesis,
purification, and characterization; spectroscopy; electrochem-
ical measurements; and calculations. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
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AUTHOR INFORMATION
Corresponding Author
ejpetersson@sas.upenn.edu
■
Present Address
†Temple University School of Medicine, Philadelphia PA
19140, United States
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(23) Mansoor, S. E.; DeWitt, M. A.; Farrens, D. L. Biochemistry 2011,
49, 9722.
(24) Batjargal, S. B.; Wang, Y. J.; Goldberg, J. M.; Wissner, R. F.;
This work was supported by funding from the University of
Pennsylvania, the National Science Foundation (CHE-1020205
to E.J.P.), and the Searle Scholars Program (10-SSP-214 to
E.J.P.). We thank Jerome Robinson and Eric Schelter for
assistance with electrochemical measurements, Jeff Saven for
Petersson, E. J. J. Am. Chem. Soc. 2012, 134, DOI: 10.1021/ja2113245.
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