Labeling proteins with fluorophore/thioamide Foerster resonant energy transfer pairs by combining unnatural amino acid mutagenesis and native chemical ligation

Article abstract of DOI:10.1021/ja4005943

We have recently shown that p-cyanophenylalanine (Cnf) and a thioamide can be used as a minimally perturbing Foerster resonant energy transfer (FRET) pair to monitor protein conformation. We have also shown that thioamide analogues of natural amino acids can be incorporated into full-sized proteins through native chemical ligation. For intermolecular studies with Cnf/thioamide FRET pairs, Cnf can be incorporated into proteins expressed in Escherichia coli through unnatural amino acid mutagenesis using a Cnf-specific tRNA synthetase. For intramolecular studies, a Cnf-labeled protein fragment can be expressed in E. coli and then ligated to a thioamide-labeled peptide synthesized on solid phase. This combination of methods allows for rapid access to double-labeled proteins with a minimum of unnecessary chemical synthesis. We demonstrate the utility of this approach by studying the binding of peptides to the protein calmodulin and by determining the orientation of the N- and C-termini in the amyloidogenic protein α-synuclein.

Full text of DOI:10.1021/ja4005943

Article
pubs.acs.org/JACS
̈
Labeling Proteins with Fluorophore/Thioamide Forster Resonant
Energy Transfer Pairs by Combining Unnatural Amino Acid
Mutagenesis and Native Chemical Ligation
Rebecca F. Wissner, Solongo Batjargal, Colin M. Fadzen, and E. James Petersson*
Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United States
S
* Supporting Information
ABSTRACT: We have recently shown that p-cyanophenyla-
lanine (Cnf) and a thioamide can be used as a minimally
perturbing Forster resonant energy transfer (FRET) pair to
̈
monitor protein conformation. We have also shown that
thioamide analogues of natural amino acids can be
incorporated into full-sized proteins through native chemical
ligation. For intermolecular studies with Cnf/thioamide FRET
pairs, Cnf can be incorporated into proteins expressed in
Escherichia coli through unnatural amino acid mutagenesis
using a Cnf-specific tRNA synthetase. For intramolecular studies, a Cnf-labeled protein fragment can be expressed in E. coli and
then ligated to a thioamide-labeled peptide synthesized on solid phase. This combination of methods allows for rapid access to
double-labeled proteins with a minimum of unnecessary chemical synthesis. We demonstrate the utility of this approach by
studying the binding of peptides to the protein calmodulin and by determining the orientation of the N- and C-termini in the
amyloidogenic protein α-synuclein.
indicating the thiocarbonyl (thioleucine is shown in Figure 1).
Distance constraints are assigned by computing quenching
efficiency (EQ = 1 − FThio/FOxo) from the fluorescence of
equimolar concentrations of Cnf/thioamide-labeled protein
(FThio) and a Cnf-labeled control (FOxo). After appropriate
INTRODUCTION
■
Fluorescence spectroscopy can be used to monitor conforma-
tional changes in proteins by exploiting distance-dependent
interactions such as Forster resonant energy transfer
̈
(FRET).1,2 In a FRET experiment, a donor fluorophore is
paired with an acceptor fluorophore or quencher. An increase
in chromophore separation leads to an increase in donor
fluorescence and a decrease in acceptor fluorescence or
quenching with an R−6 distance dependence. The utility of
the data obtained in these experiments often depends on the
ability to label the proteins at appropriate locations. Bulky
fluorophores are limited in the number of places at which the
label can be placed, and are subject to the concern that the label
itself alters the observed motion.3 Therefore, our laboratory has
developed the thioamidereplacing oxygen with sulfur in the
peptide backboneas an extremely small label compatible with
virtually any position in the protein (Figure 1).
corrections to the Forster radius (R ) for each Cnf location, the
̈
0
Cnf/thioamide separation (RFRET) is determined according to
Forster theory (Figure 1, and discussion below). These distance
̈
measurements are used to monitor conformational changes in
the protein.
All of our initial studies used small peptides and proteins in
which the thioamide was incorporated by solid-phase peptide
synthesis (SPPS). In order to use fluorophore/thioamide pairs
to monitor structural changes in full-sized proteins, we needed
to identify methods for incorporating these two modifications
with high efficiency. We have recently published an account of
our studies of the compatibility of thioamides with native
chemical ligation (NCL) reactions.7 NCL is a valuable method
for synthesizing proteins by fragment condensation of a C-
terminal thioester peptide and an N-terminal Cys peptide in
buffered aqueous media.8,9 We found that thioamides could be
placed at nearly any position in the thioester or Cys peptide
fragments. Furthermore, we found that thioamides could be
used in NCL reactions in which the N-terminal Cys fragment
was expressed as a protein construct in Escherichia coli
(expressed protein ligations). In general, we wish to minimize
the need for manual synthesis by maximizing the role of
Thioamides can quench fluorophores that have spectral
emission overlap with thioamide absorption, such as
cyanophenylalanine (Cnf or F*) and tyrosine, through a
FRET mechanism.4,5 Other fluorophores are quenched through
a photoinduced electron transfer (PET) mechanism, like
tryptophan, coumarin, and acridone.5,6 Here we will focus on
thioamide FRET partners, for which more reliable distance
information can be obtained by applying Forster theory. To use
̈
thioamide FRET quenching to study protein conformation, we
generate constructs in which we label the protein with a Cnf
donor fluorophore and a thioamide analogue of one of the
natural amino acids. We denote these analogues using either
the three or one letter amino acid code with a prime symbol
Received: January 18, 2013
© XXXX American Chemical Society
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the unnatural amino acid is purified for use in biochemical
experiments or subsequent NCL reactions (Figure 2).
Here, we made use of Uaa mutagenesis to incorporate donor
fluorophores for thioamide FRET experiments examining both
protein/protein interactions and intramolecular measurements
of protein folding. In intermolecular experiments, no
manipulation of the Uaa-containing protein is required
following purification. The protein is simply mixed with a
thioamide-labeled peptide or protein and quenching observed.
We use this strategy to examine a series of pairwise interactions
in which Cnf is incorporated in the calcium sensor protein
calmodulin (CaM) and the thioamide is incorporated in a
CaM-binding peptide. The combinatorial nature of these
experiments allows one to rapidly generate a matrix of m×n
FRET measurements from m Cnf-labeled proteins and n
thioamide-labeled peptides. To carry out intramolecular
experiments, we combined Uaa mutagenesis with NCL. Cnf
was incorporated by Uaa mutagenesis into protein constructs in
which the termini could be used in ligation to thiopeptides.
Strategies for ligating to both the C- and N-termini are
described. The resulting double-labeled proteins can then be
used in intramolecular folding experiments. Again, combinato-
rial assembly can be used to make m×n double-labeled proteins
from m N-terminal fragments and n C-terminal fragments. We
applied our double-labeling strategy to study conformational
changes in α-synuclein (αS), an amyloidogenic protein found
to aggregate in Parkinson’s disease.12−14
Figure 1. Cyanophenylalanine/thioamide FRET interactions. Top left:
Cyanophenylalanine (Cnf, F*) and leucyl thioamide (Leu′, L′). Top
right: Normalized absorption (dotted black line) and fluorescence
emission (dashed black line) spectrum of Cnf and absorption
spectrum of Leu′Ala (yellow line). The shaded area indicates the
spectral overlap that contributes to FRET. Bottom: Distance (RFRET
calculations are made using Forster theory as described in text.
)
̈
biosynthesis in preparing labeled proteins. These protein
ligations can limit the portion that must be prepared by
SPPS to a few amino acids around the thioamide. SPPS is
deemed necessary for the incorporation of backbone thioamide
modifications, but side-chain fluorophores like Cnf can be
incorporated during ribosomal biosynthesis.
To easily incorporate Cnf, we have employed unnatural
amino acid (Uaa) mutagenesis methods pioneered by Schultz
and co-workers.10,11 These methods allow one to insert a Uaa
at a specific site in the protein by using a so-called orthogonal
aminoacyl tRNA synthetase (RS) that is selective for the Uaa.
The UaaRS charges a tRNA that recognizes the UAG stop
codon. The UAG stop codon is mutated into the protein
coding sequence at the site of interest. Plasmids coding for the
UaaRS, tRNA, and protein of interest are transformed into E.
coli. After growth in the presence of the Uaa, protein containing
Our methods should be applicable to a wide variety of
proteins: essentially any proteins that express well in E. coli and
are amenable to refolding after ligation. Moreover, since this
combination of Uaa mutagenesis and NCL to label proteins on
the backbone and side chain is unprecedented (examples of
side-chain ligation using Uaa mutagenesis exist),15,16 the
considerations here should be useful to those who wish to
combine these two methods for other reasons, such as
incorporating olefin or D-amino acid modifications in
combination with unnatural side chains.17−20
RESULTS AND DISCUSSION
■
To begin combining Uaa mutagenesis with thioamides, we used
CaM as a model system. We chose CaM because it expresses
Figure 2. Combining unnatural amino acid mutagenesis and native chemical ligation. Thioamide-bearing peptides are synthesized on solid phase
starting from activated benzotriazole precursors and standard Fmoc-protected amino acids. For N-terminal thioamides, a thioester is installed as a
masked group or formed by postsynthetic activation. To generate the Cnf-labeled protein fragment, E. coli cells are transformed with two DNA
plasmids, one encoding the protein with a UAG stop codon at the site of interest, and one encoding an aminoacyl tRNA synthetase that is selective
for an unnatural amino acid (UaaRS) with a tRNA that recognizes the UAG stop codon. Protein is expressed under standard conditions and the N-
terminal Cys generated by proteolysis. Ligation of the two fragments gives the full-length protein, labeled with both Cnf and the thioamide.
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well in E. coli and it has been thoroughly studied from a
structural and biophysical standpoint.21−27 CaM has been
shown to bind a variety of helical peptides derived from its
endogenous protein interaction partners.28,29 These peptides
bind with high affinity in a central groove formed by the folding
of the N- and C-terminal CaM lobes. In particular, we studied
the binding of a peptide derived from the intracellular domain
of an olfactory cyclic nucleotide-gated ion channel
(pOCNC).30 This peptide binds with a low nanomolar affinity,
and its CaM-bound structure has been determined previously
by NMR.30,31 Therefore, we expected that 1:1 mixtures of 10
μM each CaM and pOCNC would be stably bound and that
the NMR structures could be used to determine interatomic
distances. These measurements would then be compared to the
corresponding distances derived from our FRET measure-
ments.
Since Cnf is excited at 240 nm and fluoresces with a
maximum at 295 nm, we began by mutating the two
endogenous Tyr residues in CaM to Phe, generating a
construct hereafter referred to as CaMF.32 This “all-Phe
background” ensures that Tyr fluorescence does not confound
our ability to interpret Cnf data. Both the extinction coefficient
and quantum yield of Phe (ε240 = 65 cm−1 M−1, Φ = 0.022) are
substantially smaller than the values for Cnf (ε240 = 10 333
cm−1 M−1, Φ = 0.110), so in these proteins all fluorescence
emission at 295 nm should derive from Cnf.32,33
To incorporate Cnf, we used a plasmid containing a UaaRS/
tRNA pair previously selected for Cnf incorporation by Mehl
and co-workers.34 This was paired with a plasmid encoding
CaM under isopropyl D-galactoside (IPTG) control. We
expressed CaMF in M9 minimal medium supplemented with
Cnf and purified the full-length protein by two rounds of
phenyl-sepharose (PS) chromatography. Four positions were
chosen for incorporation of Cnf: F13, F17, F93, and Y100. Four
pOCNC peptides were synthesized: an all-Phe oxopeptide
(pOCNC) and three thiopeptides, pOCNC-F′1, pOCNC-L′11,
and pOCNC-F′16. Before performing fluorescence titration
experiments, native polyacrylamide gel electrophoresis (PAGE)
analysis was used to ensure that each CaMF/pOCNC
combination formed stable complexes. This allowed us to
eliminate CaMFF*17 from further binding experiments (see
Supporting Information).
Figure 3. FRET in CaM/peptide complexes. Top: Binding of
thioamide-labeled peptides (green) to Cnf-labeled CaM (blue) in
the presence of Ca2+. Bottom left: An image showing Cnf (pink) at
position 100 in calmodulin (CaMFF*100) and thiophenylalanine
(yellow sulfur atom) at position 1 in the pOCNC peptide
(pOCNC-F′1). Ca2+ ions are shown as purple spheres. Adapted
from PDB ID 1SYD using PyMol. The 18.9 Å separation shown
represents an average of the separation of the center of the thioamide
from the center of the ε-carbons of the Cnf ring. Bottom right:
Fluorescence emission spectra of solutions of 10 μM CaMFF*100 in the
presence of increasing concentrations of pOCNC-F′1. Inset: Relative
fluorescence of complexes of CaMFF*100/pOCNC-F′1 (FThio) to an
oxoamide control CaMFF*100/pOCNC (FOxo). See Supporting
Information for details.
We completed comparable experiments for each pair of Cnf
mutant and thiopeptide (Figure 4). The separation in the seven
pairs spanned a distance range from 8 to 31 Å, covering the
range that is accessible with the Cnf/thioamide FRET pair. In
the case of CaMFF*100/pOCNC-F′1, no change in fluorescence
was observed in the “oxoamide control” experiment. For other
mutants, some changes in fluorescence were observed for
pOCNC binding (see Supporting Information). Regardless,
these effects can be deconvoluted by calculating EQ relative to
the oxoamide control as we do. Such oxoamide control
experiments are essential as many factors can influence
fluorescence.35−37 Therefore, we always compare any thioamide
quenching measurement to the most relevant control, in this
case the CaM/pOCNC oxopeptide complex.
For each of the other Cnf mutants, we performed the same
set of experiments, which are described using CaMFF*100 as an
example. Fluorescence wavelength scans were acquired for
mixtures of 10 μM CaMFF*100 with between 0 and 15 μM
pOCNC or pOCNC-F′1. For each concentration, the
The oxopeptide complex is also valuable in determining the
appropriate value of Φ to be used in FRET distance
calculations. The changes in fluorescence intensity upon
pOCNC binding represent a change in Cnf quantum yield as
fluorescence emission from the pOCNC-F′1 complex (FThio
)
was normalized to the fluorescence emission from the pOCNC
complex (FOxo). For F*100 a concentration-dependent decrease
was observed for FThio, while no change was seen FOxo. Plotting
F
Thio/FOxo (or 1 − EQ) as a function of concentration shows
that binding reaches saturation at 1:1 CaMFF*100/pOCNC-F′1
(Figure 3). The quenching (FRET) efficiency can then be used
to calculate a separation according to RFRET = R0(1/EQ − 1)1/6
We observed 25% quenching at saturating concentrations of
.
pOCNC-F′1, which translates into a 20 Å separation using
Forster theory. This is in excellent agreement with the 19 Å
̈
average separation computed from the 20 lowest energy
structures deposited from the NMR structure determination by
Contessa et al.31 These calculations were performed using Φ =
0.110 and an orientation factor (κ2) of 2/3.
Figure 4. CaM/pOCNC label sites. Cnf shown at three positions in
CaM with three possible pOCNC thiopeptides (coloring as in Figure
3). Images based on one of the 20 low-energy structures in PDB ID
1SYD, modified using PyMol.
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it moves into a new environment. For example, the emission of
CaMFF*93 starts out at 3.8% of the emission of an equimolar
concentration of CaMFF*100, but it increases 3.2-fold upon
pOCNC binding. Therefore, the relevant quantum yield for the
CaMFF*93/pOCNC complex is 0.014. We use this value in our
calculation of RFRET for all thiopeptide complexes with
CaMFF*93. Similar corrections to the quantum yield were
performed for CaMFF*13. Correction of the quantum yield of
CaMFF*100 was not necessary, presumably because Cnf is
solvent-exposed at this position.
terminal truncation that could be revealed after proteolysis of a
His10 purification sequence using Factor Xa (Figure 5A).38 The
The distances calculated from our FRET measurements are
collected in Table 1. All measurements are within 3 Å of the
Table 1. Distances in CaM/pOCNC Complexes
a
b
c
d
CaM/pOCNC mutant (Φ)
obs EQ (%)
RFRET (Å)
RNMR (Å)
all-Phe control
F*13/F′1 (0.003)
F*13/L′11 (0.003)
F*17
0
11
37
1
2
13
11
14
14
e
NB
F*93/F′1 (0.014)
F*93/L′11 (0.014)
F*100/F′1 (0.110)
F*100/L′11 (0.110)
F*100/F′16 (0.110)
79
43
25
17
10
2
4
3
2
12
13
20
21
23
9
12
19
24
31
f
3
a
Φ determined by comparison to fluorescence emission of 10 μM Cnf,
b
c
Φ = 0.110. EQ determined for 1:1 CaM/pOCNC complex. RFRET
d
calculated from EQ as described in the Supporting Information. RNMR
is an average value calculated from the 20 lowest energy structures in
e
PDB ID 1SYD. NB indicates no binding by native PAGE analysis.
f
Mixture of R17 D- and L-epimers of pOCNC-F′16 peptide. Both bind
with high affinity by native PAGE analysis.
Figure 5. Semisynthesis of double-labeled proteins with N-terminal
thioamides. Expressed C-terminal protein fragment (A) using a
plasmid encoding an N-terminal HisTag with a Factor Xa site and (B)
using a plasmid encoding the truncated protein where N-terminal Met
is removed by Met aminopeptidase. MeONH2·HCl used to cleave the
thiazolidine which forms with intracellular aldehydes (R = Me or
CH2CO2H). Synthetic N-terminal thiopeptide thioester: (C) synthesis
on Cl-Trt resin and activation to form thioester with PyBOP, followed
by deprotection with TFA, and (D) synthesis on Rink amide resin
with CbPGo masked thioester. The expressed protein fragment and
synthetic thiopeptide are combined in the presence of thiophenol, and
the ligated, full-length protein is isolated.
average value observed in the NMR structure except for F*100
/
F′16. Moreover, our measurements are always correct in
predicting the relative order of thioamide proximity for a
given Cnf mutant. While it may be surprising that we achieve
reasonable agreement between theory and experiment using a
value of 2/3 for κ2, deriving κ2 values from the 20 lowest energy
NMR structures has only a small effect on this agreement (see
Supporting Information). The RFRET values obtained using κ2 =
2/3 are more representative of the application of thioamide
FRET in a structure determination experiment (i.e., one in
which the target structure is unknown and κ2 would be difficult
to fit accurately).
These CaM experiments involved intermolecular quenching,
but our method, like FRET in general, is equally applicable to
intramolecular conformational changes. However, obtaining
Cnf/thioamide double-labeled proteins represents a biosyn-
thetic challenge, as one ideally wishes to have complete
freedom in placing the chromophores within the protein
sequence. While this is easily accomplished in small peptides
made by SPPS, and conceivably could be done by NCL, we
wished to use a combination of NCL and Uaa mutagenesis to
minimize unnecessary peptide synthesis. Therefore, we assessed
the compatibility of these two methods. We chose αS as a
model system because we had previously shown that
thioamides could be incorporated into αS, and we wished to
compare PET quenching of Trp in αS to FRET quenching of
Cnf at similar locations.7
proteolyzed protein contains an N-terminal Cys for use in NCL
with the synthetic thioester. The Factor Xa method provides a
general method for generating NCL constructs containing
unnatural amino acids, but in the specific context of αS,
prolonged incubation with Factor Xa can lead to protein loss
via aggregation. Therefore, we have also used constructs
directly encoding αS with an N-terminal truncation plus Met in
a fashion similar to that described by Hejjaoui et al.39 The N-
terminal Met residue is removed in vivo by Met aminopeptidase
to reveal the requisite Cys.40 Protein harvested from E. coli
contains an N-terminal thiazolidine ring formed by the
condensation of Cys with either pyruvate or acetaldehyde.
This is treated with methoxylamine hydrochloride to reveal free
Cys for NCL reactions. Regardless of the method used in
producing the expressed fragment, once purified, usage in
subsequent NCL reactions is identical.
First, we examined thioamide incorporation at the αS N-
terminus. For protein expression, we used the UaaRS/tRNA
plasmid pDULE2-Cnf and a plasmid encoding αS with an N-
Synthesis of a thioamide-containing thioester peptide using
9-fluorenylmethoxycarbonyl (Fmoc) chemistry could be
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accomplished by one of two methods. The thiopeptide can be
synthesized on 2-chlorotrityl (Cl-Trt) resin, cleaved under mild
conditions (10% AcOH) to retain the side-chain protecting
groups, and then activated using benzotriazol-1-yloxytripyrro-
lidinophosphonium hexafluorophosphate (PyBOP) to form the
thioester (Figure 5C).41,42 This method can lead to
epimerization of the α-carbon of the C-terminal amino acid,
but this can be avoided by using short (<30 min) reaction times
during PyBOP activation.43 A more significant inherent
limitation is the insolubility of protected peptides >15 amino
acids in most solvents.41 This limits use of the PyBOP method
to short peptides.
The other method makes use of a thioester that is masked
during synthesis as a disulfide (Figure 5D). Several variations of
masked thioester exist. In our previous work, we used a three-
residue CbPGo sequence, where Cb is a t-Bu-protected Cys (S-
tert-butyl-L-cysteine) and Go is glycolic acid.7,44,45 Deprotection
of the Cys thiol initiates a cascade reaction generating a
diketopiperazine thioester that is competent in NCL reactions.
Synthesis of peptides with the CbPGo linker is inefficient
because of ester linkage instability. Therefore we reserve its use
for longer peptides where it is necessary.
All αS protein constructs tested contain Tyr-to-Phe
mutations at positions 39, 125, 133, and 136 to generate an
all-Phe background construct, αSF. As was the case for CaMF,
these mutations allow us to more easily interpret Cnf
fluorescence data. Combinations of method 5A or 5B with
5C or 5D were used to synthesize double-labeled αS. While all
combinations were viable, for αSFF′4C9F*94, our highest yields
were obtained with a combination of methods 5B and 5C.
Protein expression yields were typically 3 mg/L of culture after
methoxylamine treatment. Ligations were typically carried out
on the 0.1 μmol scale at pH 7.2 with 1% thiophenol. After 24 h,
60−70% yields of ligated product were typically seen, and the
products purified by C4 reverse phase HPLC, giving isolated
yields of about 0.4 mg for a given ligation. PAGE,
spectroscopic, and matrix-assisted laser desorption/ionization
mass spectrometry (MALDI MS) analyses of the ligation
confirm production of homogeneous, double-labeled αS
(Figure 6). Data for other reactions are shown in the
Supporting Information.
Figure 6. Characterization of αSFF′4C9F*94 ligation product. Top left:
PAGE showing αSF9−140C9F*94 before (1) and after (2)
MeONH2·HCl treatment, ligation of αSF9−140C9F*94 to αSF1−8F′4-SR
thioester (3), purified αSF-F′4C9F*94 (4), αSFC9F*94 (5), and αSWT
(6). See HPLC chromatogram in the Supporting Information for
separation of ligated product. Top right: MALDI MS analysis of full-
length αSFF′4C9F*94. Bottom left: UV/vis absorption spectrum of
αSFF′4C9F*94 showing thioamide absorption at 266 nm. Inset: MALDI
MS analysis of trypsinized 1−6 fragment, confirming the presence of
the thioamide at F′4 [calcd m/z (M+H) 828.35, obsd 828.65]. The
asterisk indicates the absence of peaks corresponding an oxoamide at
Phe4 in the 1−6 fragment [calcd m/z (M+H) 812.38]. Bottom right:
Fluorescence emission spectra of αSFF′4C9F*94 showing Cnf emission
at 295 nm. Inset: MALDI MS analysis of trypsinized 81−96 fragment,
confirming the presence of the Cnf at position 94 [calcd m/z (M+H)
1503.78, obsd 1504.30]. The asterisk indicates the absence of peaks
corresponding Tyr at position 94 in the 81−96 fragment [calcd m/z
(M+H) 1494.78].
The methods shown in Figure 5 permit protein double-
labeling with thioamides located at the N-terminus. To insert
thioamides at the C-terminus, we made use of intein fusion
constructs popularized by Muir and others.9 These self-splicing
domains have been engineered to stall at a thioester
intermediate and can be intercepted by treatment with a
small molecule thiol to give a thioester suitable for NCL. We
have found that the expression of intein fusion constructs is
compatible with Uaa mutagenesis. In fact, yields of intein
fusions of αS Cnf mutants (e.g., αSF1−122F*39-Int) are higher
(20 mg/L culture) than comparable expressions of the full-
length protein using either T7 or PRK vectors previously used
to express αS mutants (3−5 mg/L). The synthetic thiopeptide
with N-terminal Cys (αSF123−140C123A′124) was prepared by
standard SPPS (Figure 7).
Figure 7. Semisynthesis of double-labeled proteins with C-terminal
thioamides. An expressed N-terminal protein fragment is generated
using a plasmid encoding a truncated version of the protein with a C-
terminal intein. Thiolysis of the stalled intein generates a thioester
suitable for NCL. The synthetic thiopeptide with an N-terminal Cys is
made by standard SPPS. The expressed protein fragment and synthetic
thiopeptide are combined in the presence of thiophenol, and the
ligated, full-length protein is isolated.
The thioester αSF1−122F*39-SR was prepared from
αSF1−122F*39-Int using the thiol sodium 2-mercaptoethanesul-
fonate (MESNA). This was combined with αSF123−140C123A′124
in a buffer containing thiophenol. Although some C-terminal
Asn cyclization (∼25% based on crude MALDI MS estimates;
see Supporting Information) of the intein thioester occurred in
competition with the desired MESNA thiolysis, this was not a
major problem since the resulting cyclic succinimide was not
competent for ligation. Ligations were typically carried out on
the 0.3 μmol scale. After 16 h, 50% yields were obtained, and
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the products were purified by fast protein liquid chromatog-
raphy (FPLC) using an ion-exchange column, giving isolated
yields of 0.6−0.7 mg for a given ligation reaction. Once again
PAGE, spectroscopy, and MALDI MS were used to analyze the
ligation reaction. These data are shown in Figure 8. The data
confirm that we have again produced homogeneous, full-length,
double-labeled αS.
In order to demonstrate the value of double-labeling a
protein such as αS, we carried out experiments in which
intramolecular conformational changes were monitored. αS
aggregates by first forming soluble oligomers which eventually
convert into larger, insoluble fibrils.13,14,47,48 Both the oligomers
and fibrils have been shown to be cytotoxic, and recent
evidence has shown that extracellular αS can enter neurons and
catalyze misfolding of αS within healthy neurons.49−52
Therefore, there is substantial current interest in understanding
the structural dynamics of the metastable αS monomers and
the rearrangements that occur on formation of oligomers and
subsequent fibrillization. Several researchers have recently
shown the value of being able to construct semisynthetic αS
to study the effects of post-translational modification on its
folding, our modification should provide a uniquely non-
perturbing method for tracking its dynamics.7,39,53−55
We envision thioamide quenching as a method for site-
selectively monitoring conformational changes during αS
refolding. While a number of researchers have studied αS
conformation using other techniques such as NMR, IR, and
EPR, we find fluorescence to be particularly useful because the
sensitivity of the technique permits one to use low
concentrations of double-labeled αS which should not undergo
aggregation on the time scale of our experiments.47,48,52,56−60
This strategy allows us to monitor long-range (25−30 Å)
interactions that are difficult to observe with other techniques.
Moreover, FRET is easily applied to interactions involving
residues in dynamic regions of proteins, such as the termini of
αS.
We have previously shown that conformational dynamics
during aggregation can be monitored by intramolecular
quenching of Trp by a thioamide.7 Here, we demonstrate
that we can monitor conformational changes in monomeric αS,
using urea or trimethylamine oxide (TMAO) to denature or
compact αS, respectively (Figure 9). Performing oxoamide
Figure 8. Characterization of αSFF*39C123A′124 ligation product. Top
left: PAGE showing αSF1−122F*39-Int before (1) and after thiolysis of
the intein to generate αSF1−122F*39-SR thioester (2), ligation of
αSF123−140C123A′124 to αSF1−122F*39-SR thioester (3), purified
αSFF*39C123A′124 (4), αSFF*39C123 (5), and αSF (6). Top right:
MALDI MS analysis of full-length αSFF*39C123A′124. Bottom left: UV/
vis absorption spectrum of αSFF*39C123A′124 showing thioamide
absorption at 266 nm. Inset: MALDI MS analysis of trypsinized
103−140 fragment, confirming the presence of the thioamide at A′124
[calcd m/z (M+H) 4228.69, obsd 4228.47]. The asterisk indicates the
absence of peaks corresponding to an oxoamide at Ala124 in the 103−
140 fragment [calcd m/z (M+H) 4212.72]. Bottom right:
Fluorescence emission spectrum of αSFF*39C123A′124 showing Cnf
emission at 295 nm. Inset: MALDI MS analysis of trypsinized 35−43
fragment, confirming the presence of Cnf at position 39 [calcd m/z
(M+H) 960.52, obsd 960.77]. The asterisk indicates the absence of
peaks corresponding Tyr at position 39 in the 35−43 fragment [calcd
m/z (M+H) 951.51].
Using these two methods, we should be able to label a variety
of proteins with thioamides at the N- or C-terminus, with
complete freedom in placement of the donor fluorophore.
Since we have previously shown that thioamides are compatible
with the MeONH2·HCl conditions used to deprotect N-
terminal thiazolidines, the methods used here should also be
compatible with internal thioamide-labeling through three
component ligations. In addition, we note that the use of C-
terminal inteins may be generally useful to those employing
Uaa mutagenesis. One major limitation of the method is that
the inherent competition with release factors at the UAG stop
codon can lead to protein truncation.46 Purifying the full-length
protein containing the Uaa can be difficult for sites near the C-
terminus, where the truncated form is only slightly shorter. The
His-tagged intein provides a convenient handle that will only be
present in the untruncated protein. Since the intein can be
cleaved by thiolysis or hydrolysis in a traceless manner, the
Uaa-labeled fragment or protein can be obtained in pure form
in an efficient manner.
Figure 9. Refolding assay. Monomeric double-labeled αS (e.g.,
αSFF′4C9F*94) is diluted to 1 μM in either Tris buffer with 1 mM
BME or the same buffer with urea or TMAO added. Cnf fluorescence
is measured at 295 nm (FThio). Identical experiments are carried out
with donor-only labeled αS (e.g., αSFC9F*94) to determine FOxo. EQ
=
1 − FThio/FOxo
.
control experiments allows us to correct for any changes in
fluorescence not due to the thioamide, and to correct the Cnf
quantum yield in order to properly interpret the observed
quenching in terms of interchromophore distance.
The NCL procedures that we use to synthesize our
thioamide-labeled proteins introduce a Cys into the αS
sequence at position 9 or 123. However, we have previously
shown that the presence of non-native Cys in the sequence
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does not cause aberrant aggregation when incubated with β-
mercaptoethanol (BME) to prevent disulfide formation.7 While
Cys can have an effect on Cnf emission, since we include Cys
mutations in our oxoamide control experiments, we can
account for these changes as well.34,61
We carried out refolding experiments with the constructs
described above, αSFF′4C9F*94 and αSFF*39C123A′124, as well as
αSFF′4C9F*39. In the “native” state, when our constructs were
incubated in buffer alone, we observed varying levels of
quenching relative to the equivalent oxoprotein control (Figure
10). For F*94/F′4 and F*39/A′124 we observe no quenching. For
is nonetheless an observable expansion of αS once one corrects
for inherent effects on Cnf fluorescence (see Supporting
Information). In contrast, an increase in quenching (i.e., FRET)
was observed as a function of TMAO concentration for all
three mutants. The changes in EQ as a function of TMAO
concentration can be seen in Figure 10. This indicates increased
compaction of both N- and C-termini, as well as folding of the
N-terminal region on itself.
To assign distance constraints based on these measurements,
we converted the measured E values to distances using Forster
̈
Q
theory. As with CaM, we computed a specific R0 value for each
construct using the quantum yields of Cnf for the oxoamide
control proteins αSFC9F*39 (R0 = 15.6 Å in 0 M TMAO),
αSFC9F*94 (R0 = 13.6 in Å in 0 M TMAO), or αSFF*39C123 (R0
= 13.5 Å in 0 M TMAO). Forster radii for each mutant in other
̈
TMAO concentrations are given in the Supporting Informa-
tion. We use these rather than simply using R0 values calculated
from free Cnf because the variation among the different
mutants demonstrates that the protein context influences
quantum yield. The RFRET values that result from our
calculations indicate that Cnf and the thioamide come into
very close proximity in TMAO. While these results are
consistent with the single-molecule studies of Deniz and co-
workers, it is surprising that our RFRET values are so small.64
This may be the result of a dominant contribution from very
close contact. Ferreon et al. observe subpopulations with near
unity FRET efficiencies, but the Alexa Fluor FRET pair that
they use has high FRET efficiency for any separation less than
30 Å.63,67 Our probes have a shorter effective range (8−31 Å)
than theirs, and therefore provide a more precise report on the
close contact induced by TMAO. We will continue to study this
phenomenon, not only with thioamides, but also with other
short-range FRET pairs such as Cnf and Trp.68,69 Finally, it
may be more appropriate to use polymer models rather than
Figure 10. Refolding of αS in TMAO. Top: Quenching efficiency of
αSFF′4C9F*94 (red), αSFF*39C123A′124 (green), or αSFF′4C9F*39
(blue) determined in phosphate buffer at 25 °C at varying
concentrations of TMAO. Quenching efficiency is determined by
comparison to the equivalent oxoprotein, αSFC9F*94, αSFF*39C123, or
αSFC9F*39, respectively. Bottom: Interchromophore distances com-
simple Forster theory to interpret our FRET data.70,71 These
̈
models have clear value when interpreting single-molecule
FRET data, which may have broad and asymmetric FRET
efficiency distributions. Although the value is less clear for
ensemble measurements, we will explore the interpretation of
our FRET data using models such as those used by Lee, Gray,
Rhoades, and Plaxco.62,65,66,72,73
puted using Forster theory as described in the Supporting Information.
̈
F*39/F′4, we observe some quenching, which we interpret as
resulting from local compaction of the N-terminus. All of these
observations are consistent with previous FRET experiments
performed on αS. The observed N-terminal compaction is
similar to the roughly 30 Å separation observed by Rhoades
and co-workers using Alexa Fluor 488 and Alexa Fluor 594 dyes
in single-molecule FRET experiments.62 Ferreon et al. observed
FRET efficiencies consistent with separations of ∼70 Å in
single-molecule FRET experiments with αS labeled at positions
7 and 84 with the same dye pair.63,64 This is well outside the
useful range of the Cnf/thioamide FRET pair, so it is not
surprising that we see no quenching for F*94/F′4. Our results
for F*39/F′4 are consistent with experiments carried out by Lee,
Gray, and co-workers using Trp and nitrotyrosine as a FRET
pair.65,66 Our F*39/A′124 data are consistent with reports by
Nath et al. showing a ∼45 Å interaction between probes at
positions 33 and 130, which is beyond the range of the Cnf/
thioamide pair.62
CONCLUSIONS
■
The application of our recently developed Cnf/thioamide
FRET probe pair to studies of the folding of full-sized proteins
requires the ability to synthesize those labeled proteins in an
efficient manner. Although we could, in theory, construct Cnf-
and thioamide-labeled proteins purely through ligation, we have
developed a far more expedient approach by melding the
methods of unnatural amino acid mutagenesis and protein
ligation. Intermolecular studies simply require that Cnf be
incorporated into proteins expressed in E. coli through
established unnatural amino acid mutagenesis techniques.
Intramolecular studies require more complex semisynthesis to
obtain doubly labeled proteins, wherein a Cnf-labeled protein
fragment can be expressed in E. coli and then ligated to a
synthetic thiopeptide. This combination of methods minimizes
SPPS of natural amino acid segments. The same methods can
be used to synthesize proteins labeled with other donor
fluorophores such as coumarin, or any fluorophore conjugated
to a bioorthogonal handle, such as p-azidophenylalanine.74,75 In
fact, we have identified a synthetase for the incorporation of the
fluorescent amino acid acridonylalanine (Acd), which we have
When 6 M urea was used as an additive, an increase in FThio
/
FOxo was observed for F*39/F′4, consistent with greater
unfolding of the protein. For the other two mutants, no
change in FThio/FOxo was observed, indicating that these regions
were indeed less compacted. Control experiments using Cnf
itself indicate that although an inherent change in Cnf quantum
yield plays a role in the observed change in fluorescence, there
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previously shown to be quenched by thioamides.6,76 We are
currently working to optimize yields of Acd/thioamide-labeled
proteins using methods analogous to those described here.
The doubly labeled proteins produced using these methods
can be applied to monitor protein folding and dynamics. For
example, here we have studied the structure of a CaM/peptide
complex and determined the average separation of residues in
the disordered protein αS in various buffer conditions. The
benchmarking results using CaM help to validate our method
and provide a foundation for the application of thioamide
FRET to other systems. These experiments show clearly that
meaningful interpretations of the interchromopore distances
require determining changes in the donor quantum yield. This
is easily accomplished using the oxoamide control experiments
that we always include in order to determine quenching
efficiency. Moreover, the dependence of quenching efficiency
on Cnf quantum yield supports a FRET mechanism for
quenching as opposed to Dexter or PET mechanisms, which
were difficult to discount based on previous data.4,77 The
confirmation of a FRET mechanism for Cnf/thioamide
interactions allows one to confidently interpret thioamide
can easily be separated by HPLC or FPLC. The major current
limitations of our labeling methods are that Uaa mutagenesis
expression yields can be low (3−5 mg/L), and if these are
coupled to inconsistent NCL yields, it can be difficult to obtain
sufficient quantities of protein for biophysical experiments. We
will continue methodological studies to improve both aspects
to make these methods as accessible as possible to the general
biochemical community.
EXPERIMENTAL PROCEDURES
■
General Information. Fmoc-L-4-cyanophenylalanine (Fmoc-Cnf-
OH) was purchased from Peptech (Burlington, MA). Boc-L-
thionophenylalanine-1-(6-nitro)benzotriazolide, Fmoc-Gln(Trt)-OH,
and Fmoc-Asn(Trt)-OH were purchased from Bachem (Torrance,
CA) or EMD Chemicals (Philadelphia, PA). PyBOP, Fmoc-Ala-OH,
Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-
OH, Fmoc-Met-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Phe-OH,
Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(Trt)-OH, Cl-
Trt resin, and Rink amide resin were purchased from Novabiochem
(San Diego, CA). Piperidine and 2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HBTU) were purchased
from American Bioanalytical (Natick, MA). Sigmacote, N,N-
diisopropyl ethylamine (DIPEA), thiophenol, trifluoroacetic acid
(TFA), and tris(2-carboxyethyl)phosphine hydrochloride (TCEP)
were purchased from Sigma-Aldrich (St. Louis, MO). Ni-NTA resin
was from Qiagen (Valencia, CA). E. coli BL21(DE3) cells were
purchased from Stratagene (La Jolla, CA). Sequencing-grade trypsin
was purchased from Promega (Madison, WI). Restriction-grade Factor
Xa protease was purchased from Novagen (San Diego, CA). All other
reagents were purchased from Fisher Scientific (Pittsburgh, PA).
Milli-Q filtered (18 MΩ) water was used for all solutions (Millipore,
Billerica, MA). MALDI mass spectra were collected with a Bruker
Ultraflex III MALDI-TOF-TOF mass spectrometer (Billerica, MA).
UV/vis absorbance spectra were obtained with a Hewlett-Packard
8452A diode array spectrophotometer (Agilent Technologies, Santa
Clara, CA). Fluorescence spectra were collected with a Varian Cary
Eclipse fluorescence spectrophotometer fitted with a Peltier multicell
holder (currently Agilent Technologies).
quenching using Forster’s equations for future applications.
̈
Nonetheless, it is worth noting that there was an imperfect
match of observed distances (RFRET) and predicted distances
(RNMR) that was not resolved by considering the orientation
parameter κ2. This may be because simply using the 20
structures developed in solving the NMR structure does not
fully account for intermediate geometries between these valleys
on the potential energy surface. Calculating RFRET by sampling
κ2 over extended molecular dynamics trajectories may give
improved agreement with the FRET data. Additionally,
fluorescence lifetime measurements (which are concentration-
independent) would remove the influence of small deviations in
concentration on our determination of EQ. We will explore
both of these aspects in subsequent work.
The αS experiments show how one might use our method in
an intramolecular sense. The addition of TMAO results in
compaction of monomeric αS, consistent with previous
independent observations by Deniz as well as small-angle X-
ray scattering experiments by Fink.64,78 The short effective
range of our probes allows us to obtain more precise
information about close contact of regions of the protein
than similar experiments with larger fluorophores. Moreover,
the small size of our chromophores leaves less uncertainty in
their location relative to the protein structure. Thus, the
thioamide probes provide new short-range information on
TMAO-induced conformations of αS, complementing data
obtained in single-molecule experiments. We continue to apply
fluorophore/thioamide pairs to the study of folding transitions
in αS monomers as well as to monitoring conformational
changes during aggregation.
Of course, our labeling strategy is not restricted to CaM or
αS. Any protein which can be expressed at high levels in E. coli
and reversibly unfolded should be amenable to double-labeling
using a combination of Uaa mutagenesis and NCL. This
strategy should be particularly appealing when one of the
modifications cannot be incorporated ribosomally (i.e., back-
bone modifications such as olefin isosteres). For larger dyes
that are attached post-translationally, Uaa mutagenesis at two
sites, or used in combination with Cys modification is probably
more efficient.79−82 However, even in those cases, it can be
difficult to separate labeled from unlabeled protein, whereas the
ligated products of our reactions differ substantially in size and
Peptide Synthesis. All peptides were synthesized using a manual,
Fmoc-based solid-phase procedure described previously.4,7 Thioamide
monomers were synthesized as previously described.4,83
CaM Expression and Purification. Each plasmid containing a
CaM mutant (pCaM-TAG13, pCaM-TAG17, pCaM-TAG93, or pCaM-
TAG100; see Supporting Information for plasmid construction) was
transformed into pDULE2-Cnf-containing E. coli BL21(DE3) cells.
These cells were cultured and made competent through the Hanahan
method.84 First, the pDULE2-Cnf plasmid was transformed into
BL21(DE3) cells. A colony was then picked based on streptomycin
(Strep) resistance to grow cultures of competent cells. Once pCaM
was transformed into these cells, selection was based on both
ampicillin (Amp) and Strep resistance. Single colonies were used to
inoculate 5 mL of LB medium containing Amp (100 μg/mL) and
Strep (100 μg/mL). This primary 5 mL culture was incubated at 37 °C
with shaking at 250 rpm overnight, and then added to 1 L of M9
minimal medium containing Amp and Strep at the same
concentrations. M9 minimal medium was prepared by adding the
following autoclaved solutions to 1 L of autoclaved water containing 6
g of Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, and 1 g of NH4Cl: 1 mL
of 2 M MgSO4, 1 mL of 15 mg/mL FeCl2 (in 1.0 M HCl), 1 mL of 15
mg/mL ZnCl2 (in acidified H2O), 2 mL of 10% Bacto yeast extract, 20
mL of 10% glycerol (v/v), and 1 μL of 1 M CaCl2. The 1 L secondary
culture was incubated at 37 °C with shaking at 250 rpm until the
OD600 reached 0.7−0.9. Protein expression was induced with IPTG
and 95 mg (0.5 mM) Cnf, and the culture was incubated at 37 °C for
an additional 12 h. The cells were recovered from media at 6000g for
10 min. The resulting pellet was suspended in 15 mL of CaM
resuspension buffer (50 mM 3-(N-morpholino)propanesulfonic acid,
100 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH
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7.5). Cell were lysed by sonication, alternating between 1 min of
sonication. CaCl2 was added to the sonicated lysate to a final
concentration of 20 mM prior to centrifugation for 10 min at 6000g, 4
°C. Following centrifugation, the supernatant was collected for
purification. CaM was purified from the cleared cell lysate using a
PS CL-4B column with EDTA as the eluent. A column with a total
resin bed volume of approximately 8 mL was first equilibrated with
four 10 mL washes of PS Buffer A (50 mM Tris base, 1 mM CaCl2).
The cleared cell lysate was then added to the column and collected as
it ran through to save any protein that did not bind to the column.
Next, the column was washed four times with 10 mL of PS Buffer A.
The column was then washed with four 10 mL portions of high-salt PS
Buffer B (50 mM Tris base, 0.5 M NaCl, 0.1 mM CaCl2). Finally, an
additional 10 mL wash of PS Buffer A was performed to restore the
column to a low-salt condition. CaM was eluted with 18 mL of PS
Buffer C (10 mM Tris base, 10 mM EDTA, pH 7.5. This elution
solution was then resaturated with CaCl2 to a concentration of 20 mM.
This elution solution was repurified on a second PS column using the
same procedure to reduce truncation product. For the second PS
column, rather than adding 18 mL of PS Buffer C directly to the
column, 1 mL elutions were collected and analyzed by sodium dodecyl
sulfide (SDS)-PAGE. Collection stopped when the protein was no
longer visible by gel. The elution fractions were dialyzed against CaM
buffer (140 mM KCl, 15 mM 4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid (HEPES), 6 mM CaCl2, pH 6.7) and stored at −20 °C.
Proteins were analyzed by SDS-PAGE and MALDI MS for purity.
CaM/pOCNC Binding Assays. After synthesis, HPLC purifica-
tion, and lyophilization, the various pOCNC peptides were redissolved
in CaM buffer (140 mM KCl, 10 mM HEPES, 6 mM CaCl2, pH 6.7).
The concentrations of the peptides were determined by UV/vis
spectroscopy, using an extinction coefficient of 10 270 M−1 cm−1 at
273 nm for the thioamide-containing peptides and 600 M−1 cm−1 at
257 nm for the oxoamide-containing peptide. The concentration of the
CaM proteins was determined through the bicinchioninic acid (BCA)
assay. For each peptide/protein combination, six solutions were
prepared that contained 10 μM CaM protein and 0−15 μM pOCNC
peptide. In addition, a solution of 10 μM peptide in the absence of
protein was made to confirm that no background fluorescence resulted
from the peptide. The solutions were prepared three times in order to
obtain data in triplicate. EQ was computed from the raw fluorescence
data for CaM with thioamide-labeled pOCNC (FThio) or pOCNC
(FOxo).
C-Terminal Fragment Expression (Factor Xa). The plasmid
pET16b_HTag-αSFΔ1−8C9F*39 (see Supporting Information for
plasmid construction) was transformed into E. coli BL21(DE3) cells
containing pDULE2-Cnf (see above), and colonies were selected on
the basis of Amp and Strep resistance. Following growth of a primary
culture in LB medium, 1 L of M9 minimal medium containing glycerol
as a carbon source (0.25% v/v) was inoculated and allowed to grow at
37 °C with shaking at 250 rpm. When the OD600 reached 0.8, Cnf was
added (150 mg), and the culture was allowed to incubate overnight at
37 °C. The boiled and cleared lysate was prepared as described and
incubated with 2 mL of Ni-NTA resin for 1 h at room temperature.7
The resin was washed with 20 mL of 50 mM Tris, 150 mM NaCl, pH
8. The protein was eluted in eight 1.5 mL portions of 50 mM Tris, 150
mM NaCl, 250 mM imidazole, pH 8. Elution fractions were analyzed
by SDS-PAGE, combined, and dialyzed against Factor Xa cleavage
buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 8.0) overnight.
Following dialysis, His-tag cleavage was achieved by overnight
Supporting Information for plasmid construction) were transformed
into competent E. coli BL21(DE3) cells with pDULE2-Cnf (see
above). Transformed cells were selected on the basis of Amp and Strep
resistance. Single colonies were used to inoculate 5 mL of LB medium
supplemented with Amp (100 ug/mL) and Strep (100 ug/mL). The
primary 5 mL culture was incubated at 37 °C with shaking at 250 rpm
for 5 h and then used to inoculate 1 L of LB medium. When the OD600
of the secondary culture reached 0.8, the cells were isolated by
centrifugation at 5000g for 15 min and gently resuspended in 1 L of
M9 minimal medium containing Cnf (100 mg). M9 minimal medium
was prepared as described above. The cells were harvested at 5000g for
15 min, and the resulting pellet was resuspended in 20 mM Tris, pH 8.
Following sonication, the cell lysate was boiled for 20 min prior to
centrifugation for 20 min at 30000g, 4 °C. The cleared lysate was
loaded onto a Superdex 200 column (25 cm) connected to a BioCad
Sprint (FPLC) system and eluted with 20 mM Tris, pH 8. FPLC
fractions from size-exclusion chromatography were analyzed by SDS-
PAGE. The fractions containing αS were then loaded onto a HighTrap
Q HP column and eluted over a 100 min NaCl gradient (0 to 0.5 M
NaCl in 20 mM Tris, pH 8). FPLC fractions from ion-exchange
chromatography were dialyzed against 20 mM Tris, 100 mM NaCl,
pH 7.4, and analyzed by SDS-PAGE, MALDI MS, UV/vis, and
fluorescence spectroscopy. Following dialysis, guanidinium hydro-
chloride and methoxylamine hydrochloride were added to a final
concentration of 6 M and 400 mM, respectively. The pH was adjusted
to 4.0 with NaOH, and the solution was stirred overnight at 4 °C.
Removal of N-terminal Cys adducts was monitored by MALDI MS.
Following deprotection, the solution was dialyzed against water and
dried in a vacuum centrifuge.
Thioester Synthesis (PyBOP). Peptide synthesis and selective
cleavage from Cl-Trt resin was performed as previously described.7
Cleaved N-terminal peptide was dissolved in THF, and 2-
mercaptopropionate (3 equiv) was added to the solution. After
stirring for 5 min, DIPEA (6 equiv) and a solution of PyBOP pre-
dissolved in warm THF (3 equiv) were added to the reaction mixture.
The solution was allowed to stir for 30 min at room temperature, at
which point the solvent was removed by rotary evaporation. Side-chain
protecting groups were removed by incubating the peptide for 10 min
with a cleavage cocktail containing TFA, triisopropylsilane, thioanisole,
and 1,2-ethanedithiol (47:1:1:1). Following concentration of the
cleavage solution by rotary evaporation, the peptide was precipitated
with ether and isolated by centrifugation. The peptide thioester was
purified by HPLC and dried in a vacuum centrifuge.
Thioester Synthesis (CbPGo). Ac-MDV′3FMKGLCbPGo
(αS1−8V′3-CbPGo) was synthesized essentially as previously described,
with the following modifications.7 Bromoacetic acid (0.0695 g, 500
μmol, 5 equiv) was preactivated with diisopropyl carbodiimide (78 μL,
500 μmol, 5.0 equiv) for 30 min in dry DMF (6 mL). Also, the peptide
(αS1−8V′3-CbPGo) was cleaved from the resin by treatment with
CH2Cl2/TFA/thioanisole (38:1:1 v/v, 5 mL) on a rotisserie for 1 h.
The resulting solution was concentrated by rotatory evaporation,
precipitated in cold diethyl ether (20 mL), centrifuged, and decanted
with ether. The precipitated peptide was dissolved in acetonitrile/
water (50:50 v/v) for HPLC purification.
Ligation To Form αSFF′4C9F*39, αSFV′3C9F*39, and
αSFF′4C9F*94. The expressed C-terminal protein fragment (1 equiv,
0.1 μmol) was dissolved in 100 μL of freshly prepared ligation buffer
(6 M Gdn·HCl, 200 mM Na2HPO4, 20 mM TCEP, 1% v/v
thiophenol, pH 7.2 for PyBOP-based thioesters or pH 8.0 for CbPGo
thioesters). The dissolved protein fragment was transferred to a
microcentrifuge tube containing 1.2 equiv of the dried N-terminal
peptide thioester, purged with argon, and allowed to incubate
overnight at 37 °C with shaking at 1000 rpm. The ligation solution
was diluted to 3 mL and dialyzed against water prior to HPLC
purification (see Supporting Information). Following HPLC purifica-
tion, the product fractions were combined, concentrated, and
exchanged into 20 mM Tris, 100 mM NaCl, pH 7.4, using an Amicon
(Millipore) Ultra 0.5 mL 3 kDa spin column.
incubation with Factor Xa protease (10 units per 1 mg of HTag
-
αSFΔ1−8C9F*39). Factor Xa was inactivated by boiling the reaction for
20 min, and the precipitated enzyme was removed by centrifugation
for 20 min at 30000g, 4 °C. The supernatant was then subjected to
incubation with 1 mL of Ni-NTA resin for 1 h at room temperature to
capture any remaining HTag-αS9−140FC9F*39. The flow-through
containing αSFΔ1−8C9F*39 was collected, analyzed by SDS-PAGE
and MALDI MS, dialyzed overnight against water, and stored at −80
°C.
C-Terminal Fragment Expression (Met Aminopeptidase).
Expression and Purification of αSF1−122F*39-Int. A plasmid
encoding αS1−122F*39-Int-HTag was transformed into E. coli BL21-
The plasmids pRK_αSFΔ2−8C9F*39 and pRK_αSFΔ2−8C9F*94 (see
I
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(DE3) cells containing the pDULE2-Cnf gene (prepared and made
competent using the Hanahan method).84 A starter culture of 4 mL of
LB medium was inoculated with a single colony and was grown at 37
°C in the presence of Amp (100 mg/L) and Strep (100 mg/L) for
approximately 4 h. A secondary culture of 1 L of M9 medium was
inoculated with 4 mL of the starter culture and grown at 37 °C with
the same concentrations of Amp and Strep. M9 minimal medium was
prepared as described above. The culture was induced by the addition
of Cnf (150 mg) followed by 0.5 mM of IPTG when it reached an
OD600 of 0.8 and allowed to grow at 37 °C overnight. Cells were
harvested at 5000 rpm using a GS3 rotor and Sorvall RC-5 centrifuge
for 20 min at 4 °C. After discarding the supernatant, the pellet was
resuspended in 30 mL of resuspension buffer: 40 mM Tris, 5 mM
EDTA, pH 8.3, protease inhibitor cocktail, 1 mM phenylmethane-
sulfonyl fluoride (PMSF), and 10 units/mL DNase1-Grade II. The
cells were lysed by sonication, and the lysate was centrifuged at 13, 900
rpm for 30 min at 4 °C. The supernatant was then incubated with Ni-
NTA resin for 1 h on ice. This slurry was loaded into a column and the
liquid allowed to flow through. The resin was washed extensively, first
with 15 mL of Wash Buffer A (50 mM HEPES, pH 7.0), then twice
with 10 mL of Wash Buffer B (50 mM HEPES, 10 mM imidazole, pH
7.0. The protein was eluted with five 2 mL portions of elution buffer
(50 mM HEPES, 300 mM imidazole, pH 7.0). The eluted fractions
were combined and incubated with 400 mM MESNA for thiolysis for
48 h, stirring at 4 °C. The protein thioester was dialyzed against 20
mM Tris, pH 8.0 overnight, and incubated with Ni-NTA resin for 1 h
on ice. The flow-through was collected, dialyzed against 20 mM
sodium citrate, pH 5.0, purified over HiTrap SP column using a 100
min NaCl gradient (0 to 0.5 M NaCl in sodium citrate, pH 5). The
product fractions were pooled and dialyzed against Milli-Q water
overnight, dried in a vacuum centrifuge, and stored at −80 °C until
further use.
ASSOCIATED CONTENT
■
S
* Supporting Information
Characterization of all peptides and proteins, assessment of
CaM mutants by native PAGE and circular dichroism, primary
fluorescence data for CaM experiments, analysis of fluorescence
data using Forster theory, HPLC analysis of NCL reactions,
̈
details of UV/vis, fluorescence, and trypsin/MALDI MS
characterization of NCL products, primary fluorescence data,
and PAGE analysis of αS refolding experiments. This material is
available free of charge via the Internet at http://pubs.acs.org.
Primary data used to generate figures and tables have been
digitally archived and can be obtained upon request from the
corresponding author.
AUTHOR INFORMATION
■
Corresponding Author
ejpetersson@sas.upenn.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by funding from the University of
Pennsylvania, including a grant from the Institute on Aging, the
Searle Scholars Program (10-SSP-214 to E.J.P.), the National
Science Foundation (NSF CHE-1150351), and the National
Institutes of Health (NIH NS081033 to E.J.P.). Instruments
supported by the NSF and NIH include HRMS (NIH RR-
023444) and MALDI-MS (NSF MRI-0820996). We thank
Prof. Ryan Mehl for the gift of the pDULE2-Cnf plasmid. We
also thank Dr. Christopher Lanci of the UPenn Biological
Chemistry Resource Center for assistance with circular
dichroism spectroscopy (supported by UPenn Laboratory for
Research on the Structure of Matter with NSF MRSEC DMR
0520020, 1120901). R.F.W. thanks the NIH for funding
through the Chemistry-Biology Interface Training Program
(T32 GM07133). C.M.F. thanks the Roy and Diana Vagelos
Scholars Program in the Molecular Life Sciences for support.
Ligation To Form αSFF*94C123A′124. Protein thioester
αSF1−122F*39 (1.5 equiv, 0.45 μmol, 1.5 mM) and C-terminal protein
fragment αSF123−140C123A124 (1 equiv, 0.3 μmol, 1 mM) were dissolved
́
in 300 μL of a freshly made, degassed ligation buffer (6 M Gdn·HCl,
200 mM Na2HPO4, 20 mM TCEP, and 1% v/v thiophenol, pH 7.0).
The reaction solution was placed in an incubator at 37 °C, shaking at
1000 rpm. The ligation progress was monitored by MALDI MS, and
allowed to react until all the protein thioester was either consumed or
hydrolyzed. Upon completion, the ligation solution was brought up to
1.5 mL with water and dialyzed against 20 mM Tris, pH 8.0 for 4 h.
Then, the ligated product, αSFF*39C123A′124, was purified over a
HiTrap Q HP column using a 100 min NaCl gradient (0 to 0.5 M
NaCl in 20 mM Tris, pH 8). The product fractions were pooled,
concentrated, and the buffer was exchanged for 20 mM Tris, 100 mM
NaCl, pH 7.4, using an Amicon (Millipore) Ultra 0.5 mL 10 kDa spin
column.
Trypsin Digest. Proteins (10−50 μg) were incubated with 5−10
μL aliquots of sequencing-grade modified trypsin (0.1 mg/mL) in 20
mM Tris, 100 mM NaCl, pH 7.5, at 37 °C for 4−8 h. An aliquot (1.0
μL) of the digestion reaction was taken and analyzed by MALDI MS
(Figures 6 and 8, and Supporting Information).
Refolding Assays. Prior to performing the experiment, the
concentrations of all double-labeled proteins were adjusted using the
BCA assay to match their respective oxoamide (donor only) control
proteins. Tris buffers (20 mM Tris, 100 mM NaCl, 1 mM βME, pH
7.5) containing TMAO were prepared such that, upon addition of
protein (to a final concentration of 1 μM), the final TMAO
concentrations were 0, 1, 2, 3, and 4 M. Buffers containing urea
were similarly prepared such that, upon addition of protein, the final
urea concentration was 6 M. Fluorescence spectra were measured
immediately following sample dilution in TMAO or urea buffer (to a
final volume of 130 μL). All samples were prepared and measured in
triplicate.
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