Thioamide quenching of intrinsic protein fluorescence

Article abstract of DOI:10.1039/c1cc14708k

Thioamides quench tryptophan and tyrosine fluorescence in a distance-dependent manner and thus can be used to monitor the binding of thioamide-containing peptides to proteins. Since thioamide analogs of the natural amino acids can be synthetically incorporated into peptides, they can function as minimally-perturbing probes of protein/peptide interactions.

Full text of DOI:10.1039/c1cc14708k

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Thioamide quenching of intrinsic protein fluorescencewz
Jacob M. Goldberg, Rebecca F. Wissner, Alyssa M. Klein and E. James Petersson*
Received 31st July 2011, Accepted 25th August 2011
DOI: 10.1039/c1cc14708k
Thioamides quench tryptophan and tyrosine fluorescence in a
distance-dependent manner and thus can be used to monitor the
binding of thioamide-containing peptides to proteins. Since
thioamide analogs of the natural amino acids can be syntheti-
cally incorporated into peptides, they can function as minimally-
perturbing probes of protein/peptide interactions.
Although electron-transfer-induced quenching of Trp and
Tyr fluorescence by many functional groups is well documented,
there are only a few reports of the effect of a thioamide on
protein fluorescence.6 Wiczk et al. examined FRET quenching
of Trp in neat propylene glycol, but since thioamide absor-
bance is red-shifted and Trp emission is blueshifted under
these conditions, it is not clear that their calculations are
appropriate for aqueous solutions.7 Also, we note a report
Intrinsic protein fluorescence, which chiefly arises from excita-
tion of tryptophan (Trp) or tyrosine (Tyr) residues, has been
used extensively to probe protein folding, conformational
rearrangement, and ligand binding.1 Many of these studies
employ distance-dependent quenching of Trp and Tyr through
energy transfer to an extrinsic probe, but data interpretation in
these experiments is limited by the size of the acceptor
chromophore.2 If a chromophore were sufficiently compact
to be incorporable at any position in a protein without
severely perturbing native structure, the structural resolution
of these experiments could be greatly improved. Here we show
that a thioamide–a single atom substitution of the peptide
bond–is such a probe and can quench Trp and Tyr fluores-
cence in a distance-dependent fashion. We also show that this
technique can be used to monitor protein interactions in vitro.
We have previously demonstrated that thioamide quenching
of p-cyanophenylalanine (Cnf) can be used to study protein
dynamics.3 Cnf was chosen for these initial experiments
because of its large extinction coefficient (e = 13000 Mꢀ1 cmꢀ1
at 240 nm) and substantial spectral overlap with thioamides.4
However, the Cnf/thioamide pair is not generally compatible
by Rownicka et al. in which thiouracil, which contains a
´
thioamide, is shown to quench intrinsic fluorescence of bovine
serum albumin upon binding.8
We began our investigation by considering a possible FRET
mechanism for quenching.9 According to Forster theory, the
¨
efficiency of energy transfer between a fluorophore and
quencher depends on the spectral overlap of donor emission
and acceptor absorbance. Although the Tyr emission spectrum
has moderate overlap with the absorbance of a thioamide, the
Trp emission spectrum has almost no overlap (Fig. 1). These
observations are borne out in the theoretical calculations of
R0, the distance at which energy transfer is 50% efficient.
Using the previously reported quantum yields for Tyr (F =
0.14) and Trp (F = 0.13), and assuming the transition
moment dipoles to be randomly oriented during the course
of energy transfer (k2 = 2/3), we calculated R0 to be 13.4 A for
the Tyr/thioamide FRET pair, and 4.0 A for the Trp/thioamide
with Trp- and Tyr-containing proteins because Forster reso-
¨
nant energy transfer (FRET) between the residues can com-
plicate data interpretation.5 Since global replacement of these
aromatic residues is undesirable, we chose to explore thioa-
mide quenching of Trp and Tyr fluorescence in order to
understand the effects of thioamide incorporation on the
near-UV fluorescence of typical proteins.
Department of Chemistry, University of Pennsylvania, 231 South 34th
Street, Philadelphia, Pennsylvania 19104-6323, USA.
E-mail: ejpetersson@sas.upenn.edu; Fax: +1-215-573-2112;
Tel: +1-215-746-2221
w This article is part of the ChemComm ‘Emerging Investigators 2012’
themed issue.
z Electronic supplementary information (ESI) available: Descriptions
of peptide and protein preparation, purification, and characterization;
fluorescence and UV/Vis spectroscopy; molecular dynamics simula-
tions; and calculations. See DOI: 10.1039/c1cc14708k
Fig. 1 Thioleucylalanine ester (Leu’Ala), tyrosine (Tyr) and trypto-
phan (Trp) spectra. Absorption spectrum of Leu’Ala (solid orange
line) shown with absorption intensity normalized to extinction coeffi-
cient (12 400 Mꢀ1 cmꢀ1 at 266 nm). The fluorescence spectra of Tyr
and Trp (dashed blue and dotted red lines, respectively) are normal-
ized to emission maxima. The shaded area indicates the spectral
overlap that contributes to FRET between a thioamide and Tyr.
c
1550 Chem. Commun., 2012, 48, 1550–1552
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14
pair in aqueous solutions (see the ESI for calculationsz).10 For
small values of R0, such as those that we calculated for Trp,
N-terminus of the peptide (pOCNC-F1 ). As a control for
any changes in fluorescence due to factors other than thioamide
quenching, we prepared an oxoamide version of the peptide
(pOCNC) with phenylalanine at the N-terminus. From NMR
structural data, we estimated the distance between the
0
Forster theory may not provide an adequate prediction of
¨
quenching, since other short-range mechanisms will contribute
significantly.11
To verify the distance dependence predicted by Forster
¨
thiocarbonyl carbon and the center of the phenol ring to be
14
theory, we synthesized groups of peptides that contained either
Trp or Tyr at the C-terminus and thioleucine (Leu’) at the
N-terminus, separated by an increasing number of proline
residues.12 Fluorescence spectra of dilute samples of each
peptide were taken in phosphate buffer at pH 7.00. In both
cases, the fluorescence intensity showed a strong dependence
on distance, at length scales much greater than that predicted
19 A for Y100 and 16 A for Y139
.
Titrations of each peptide
into CaM solutions are shown in Fig. 3.
A 28% decrease in fluo0rescence is seen at saturating con-
centrations of pOCNC-F1 , whereas essentially no change in
fluorescence is seen with the oxoamide control pOCNC. Since
these peptides differ only in the O-to-S substitution in the F1/
R2 amide bond, we assign the quenching to the Tyr/thioamide
interaction. Both Trp and Tyr fluorescence are sensitive to
their local environment with respect to solvent accessibility,
pH, neighboring residues, and other chromophores.15 CaM
undergoes a substantial conformational rearrangement upon
peptide binding, and we were concerned that these factors
might alter Tyr fluorescence independent of thioamide
quenching.16,17 Therefore, inclusion of an oxoamide control
is essential to assign quenching specifically to the Tyr/thio-
by Forster theory (Fig. 2). This is particularly noticeable for
¨
Trp, where a half-maximal change in fluorescence is observed
at 17 A, far exceeding the predicted R0 of 4.0 A. The distance
dependence of both Tyr and Trp are shown fit to a sigmoidal
expression, which provides a reasonable description of the
distance dependence without assigning a mechanistic inter-
pretation. Fits of the data to other mechanism-based
equations, including Forster, Dexter, and electron transfer are
provided in the ESI,z with accompanying discussion. The
sigmoidal fit in Fig. 2 serves as a working, empirical ‘‘ruler’’
based on quenching efficiency to be used while we investigate
the mechanism of quenching further.
0
amide interaction. Corrected pOCNC-F1 fluorescence can be
fit to a 1 : 1 binding model to obtain a KD of 280 nM.y
Prediction of the efficiency of quenching (E ) from Forster
¨
Q
theory is complicated by the fact that the two donor Tyr have
different extinction coefficients (e) and quantum yields (F),
both of which contribute to FRET (i.e. quenching)
efficiency.16,18 Using reported e and F values for each Tyr, we
To test the utility of our spectroscopic ruler and verify that
quenching takes place in a molecular context other than
the Pro series, we conducted protein-binding studies in which
the thioamide was incorporated into peptides that bind to the
protein calmodulin (CaM). Since the peptides bind with a well-
defined geometry, this experiment allowed us to examine
quenching in an intermolecular context and to evaluate the
ability of our model to predict observed quenching efficiencies
in proteins with multiple fluorescent residues.
calculated EQ values from Forster theory and an empirical
¨
calibration based on Fig. 2 (see ESIz for calculations). Since
Phe is not noticeably fluorescent at the excitation wavelength
of Tyr, we generated Y100F and Y139F mutants to verify the
effect of thioamide quenching on each Tyr residue indepen-
dently. Table 1 summarizes the observed quench0ing of each
mutant in a stoichiometric ratio with pOCNC-F1 , as well as
theoretical predictions from Forster theory (E Forster) and
Chicken CaM contains two tyrosine residues, Y100 and Y139
,
and, in the presence of Ca2+, binds helical peptides with
diverse sequences.13 We prepared derivatives of bOCNCp, a
fragment of an olfactory cyclic nucleotide-gated channel, in
which thiophenylalanine (Phe’) was incorporated at the
¨
¨
Q
our empirical spectroscopic ruler (EQ Calculated) using
Fig. 3 CaM Binding pOCNC. Left: CaM structure taken from PDB
1SY9.14 Image created in PyMOL. (Delano Scientific, LLC; South San
Francisco, CA, USA) Y100 and Y139 are rendered in grey; the
thioamide bond is highlighted in yellow. Right: Titration of pOCNC
(open squares) and pOCNC-F10 (filled circles) into a solution of 10 mM
CaM in 15 mM HEPES buffer, 140 mM KCl, and 6 mM CaCl2,
pH 6.70, monitored by fluorescence spectroscopy. Fluorescence data
are normalized to CaM alone and corrected for peptide fluorescence.
Fig. 2 Fluorescence intensity as a function of chromophore separa-
tion. Left: The fluorescence emission at 305 nm of Leu’-Pron-Tyr (n =
2–10) is shown. Right: Fluorescent emission at 355 nm of Leu’-Pron-
Trp (n = 2–11). In both plots, the ‘‘N’’ data point indicates the
fluorescence of Leu-Pro2-Tyr or Leu-Pro2-Trp. Sigmoidal fits to the
data shown (3 or more trials per peptide, bars represent standard
error). Distances determined from molecular dynamics simulations.
c
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Chem. Commun., 2012, 48, 1550–1552 1551

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Table 1 Quenching of CaM fluorescence by thiopeptide
RFW thanks the NIH for funding through the Chemistry-
Biology Interface Training Program (T32 GM07133). AMK
thanks the NSF for a Summer REU Fellowship (DMR05-
20020). We thank Jeff Saven for use of the fluorometer, Feng
Gai for assistance with the CD spectrometer (supported by NSF
DMR05-20020), Rakesh Kohli for assistance with MALDI-MS
(supported by NSF MRI-0820996), and Joshua Wand for the
calmodulin plasmid.
CaM mutant
EQ (%)
EQ (%)
EQ (%)
Forsterb,c
¨
(donor, DQ distance)a experimentalb calculatedb,c
WT
Y100F (Y139, 16 A)
28 ꢁ 1
50 ꢁ 2
42
56
30
34
40
10
14
0
Y100W (W100, 19 A) 27 ꢁ 3
Y139F (Y100, 19 A)
Y139W (W139, 17 A) 58 ꢁ 2
22 ꢁ 2
8
0
a
Donor/quencher (DQ) distance measured from aromatic ring of Tyr
b
or Trp to thiocarbonyl carbon. Calculations described in ESI.z
c
Notes and references
Error estimates: Tyr: ꢁ15%; Trp: ꢁ20%.
y We note that the concentrations of peptide and protein used here
(low mM) are not optimal for determining the KD for such a high
affinity interaction.
distances from the NMR structure. There is reasonable agree-
ment between our empirical EQ and the observed EQ for the
single Tyr mutants, but the WT values deviate, perhaps
because we are not weighting the fluorophore contributions
or orientation effects (k2) properly.
1 (a) J. M. Beechem and L. Brand, Annu. Rev. Biochem., 1985, 54,
43–71; (b) M. P. Brown and C. Royer, Curr. Opin. Biotechnol.,
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127–205.
To further examine quenching of Trp, we mutated either Y100
or Y139 to Trp. Stimulation at 295 nm allowed us to selectively
excite the Trp residue. The observed EQ of the Trp mutants at
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0
stoichiometric pOCNC-F1 are reported in Table 1 along with
predicted quenching efficiencies. Several observations are worth
noting from these data. First, given the predicted and observed
distance dependencies, a FRET mechanism is clearly not responsible
for Trp quenching, and is at best partially responsible for Tyr
quenching. Second, the exponential distance-dependence one
might expect for through-bond electron transfer is seen both in
the Pro series and the CaM-binding experiments, which shows
that the rigidity of the Pro s-bond framework is not necessary
for efficient transfer over 15–20 A distances. Third, the
observed quenching efficiency in the CaM binding experiments
is in moderate agreement with the quenching level predicted from
our polyproline rulers, indicating that they may be useful as
empirical standards. Further investigation will be required to gain
a greater understanding of environment and orientation effects.
In conclusion, we have demonstrated that thioamides
quench Trp and Tyr fluorescence in a distance-dependent
fashion that can be used to monitor biological interactions,
such as macromolecular binding events. It is well established
that Trp and Tyr are also quenched by many other functional
groups. However, since we can compare our synthetic thioa-
mide proteins to all-oxoamide equivalents, we should be able
to separate thioamide-quenching from quenching by protein
sidechains or backbone in interpreting our data. We are
currently uncertain of the mechanism of quenching, but recent
data collected in our laboratory suggest that thioamides are
capable of quenching red-shifted fluorophores with no spectral
overlap such as fluorescein, implicating an electron transfer
process in quenching. Given the substantial difference in the
oxidation potentials between oxoamides (3.25 eV) and thio-
amides (1.21 eV), it is not surprising that thioamide-specific
quenching can occur.19 We are investigating this phenomenon
further in the context of Tyr and Trp as well as other
fluorophores. We are also developing methods for the synth-
esis of large thioamide-containing proteins so that the findings
here may be extended to interactions of full proteins.
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This work was supported by funding from the University of
Pennsylvania, the National Science Foundation (CHE-1020205
to EJP), and the Searle Scholars Program (10-SSP-214 to EJP).
c
1552 Chem. Commun., 2012, 48, 1550–1552
This journal is The Royal Society of Chemistry 2012