Native chemical ligation of thioamide-containing peptides: Development and application to the synthesis of labeledα-synuclein for misfolding studies

Article abstract of DOI:10.1021/ja2113245

Thioamide modifications of the peptide backbone are used to perturb secondary structure, to inhibit proteolysis, as photoswitches, and as spectroscopic labels. Thus far, their incorporation has been confined to single peptides synthesized on solid phase. We have generated thioamides in C-terminal thioesters or N-terminal Cys fragments and examined their compatibility with native chemical ligation conditions. Most sequence variants can be coupled in good yields with either TCEP or DTT as the reductant, though some byproducts are observed with prolonged TCEP incubations. Furthermore, we find that thioamides are compatible with thiazolidine protection of an N-terminal Cys, so that multiple ligations can be used to construct larger proteins. Since the acid-lability of the thioamide prohibits on-resin thioester synthesis using Boc chemistry, we devised a method for the synthesis of thioamide peptides with a masked C-terminal thioester that is revealed in situ. Finally, we have shown that thioamidous peptides can be coupled to expressed protein fragments to generate large proteins with backbone thioamide labels by synthesizing labeled versions of the amyloid protein α-synuclein for protein folding studies. In a proof-of-principle experiment, we demonstrated that quenching of fluorescence by thioamides can be used to track conformational changes during aggregation of labeled α-synuclein.

Full text of DOI:10.1021/ja2113245

Article
pubs.acs.org/JACS
Native Chemical Ligation of Thioamide-Containing Peptides:
Development and Application to the Synthesis of Labeled α-
Synuclein for Misfolding Studies
Solongo Batjargal, Yanxin J. Wang, Jacob M. Goldberg, Rebecca F. Wissner, and E. James Petersson*
Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United States
S
* Supporting Information
ABSTRACT: Thioamide modifications of the peptide back-
bone are used to perturb secondary structure, to inhibit
proteolysis, as photoswitches, and as spectroscopic labels. Thus
far, their incorporation has been confined to single peptides
synthesized on solid phase. We have generated thioamides in
C-terminal thioesters or N-terminal Cys fragments and
examined their compatibility with native chemical ligation
conditions. Most sequence variants can be coupled in good
yields with either TCEP or DTT as the reductant, though some byproducts are observed with prolonged TCEP incubations.
Furthermore, we find that thioamides are compatible with thiazolidine protection of an N-terminal Cys, so that multiple ligations
can be used to construct larger proteins. Since the acid-lability of the thioamide prohibits on-resin thioester synthesis using Boc
chemistry, we devised a method for the synthesis of thioamide peptides with a masked C-terminal thioester that is revealed in
situ. Finally, we have shown that thioamidous peptides can be coupled to expressed protein fragments to generate large proteins
with backbone thioamide labels by synthesizing labeled versions of the amyloid protein α-synuclein for protein folding studies. In
a proof-of-principle experiment, we demonstrated that quenching of fluorescence by thioamides can be used to track
conformational changes during aggregation of labeled α-synuclein.
degradation.24,25 Despite these differences, the thioamide is
generally stable at physiological pH and can be incorporated in
INTRODUCTION
The complex folding and function of proteins is dictated by the
interplay of a large number of intra- and intermolecular
noncovalent interactions. The interactions of amino acid side
■
an α-helix or a β-turn without grossly perturbing secondary
structure.26−28
Thioamide substitutions can also be used as unique
chains are probed through mutation of a given residue to one of
photochemical probes because the O-to-S substitution in the
the other 19 natural amino acids, or by replacement with an
carbonyl causes a red-shift of the absorption spectrum. This is
unnatural amino acid through ribosomal incorporation or semi-
synthesis.1,2 The role of the protein backbone can only be
manifested as circular dichroism bands at 280 and 330 nm that
can be used to monitor local conformational changes at the
rationally probed by synthetic modification, as the natural
amino acids all possess an oxoamide. A great number of
thioamide.13 The shift of the π→π* absorption from 200 nm
for the oxoamide to 260 nm for the thioamide allows one to
specifically excite the thiopeptide unit. Irradiation with 260 nm
light drives the thiopeptide bond from a predominantly trans
backbone analogues have been developed, including isosteric
ester, thioester, and thioamide substitutions.3−10 All of these
modifications can be used to probe backbone electrostatic and
hydrogen-bonding interactions, but thioamides can also be used
conformation to one with a significant cis population, making
the thiopeptide a photoswitch.12,29 Our own research has
to confer proteolytic stability, to photoisomerize the backbone,
and as a spectroscopic label in circular dichroism studies.11−13
shown that the thioamide bond can act as a quencher of p-
cyanophenylalanine (Cnf, F*), tyrosine, and tryptophan
Very recently, we have demonstrated that they also function as
fluorescence quenching probes to study protein dynamics.14,15
fluorescence, and that this quenching can be used to monitor
protein dynamics.14,15 Since analogues of the 20 natural amino
Thioamides are nearly isosteric with the natural oxoamides
acids can conceivably be inserted into a peptide sequence,
found in the peptide backbone, but there are some subtle
photochemical applications of the thioamide should have no
differences. Sulfur has a larger van der Waals radius than oxygen
inherent positional restrictions.
One particularly exciting area of application for thioamides is
the study of amyloid protein misfolding in neurological disease,
such as the Aβ peptide in Alzheimer’s disease or α-synuclein
(1.85 vs 1.40 Å), and the thiocarbonyl bond is somewhat longer
than the oxocarbonyl bond (∼1.60 vs ∼1.25 Å).16−19 The
thioamide NH is a stronger hydrogen bond donor than the
oxoamide NH, while the sulfur is a slightly weaker hydrogen
bond acceptor than the corresponding oxygen.20−23 The
thioamide sulfur is also more reactive as a nucleophile,
reactivity commonly observed in cyclization during Edman
Received: December 3, 2011
Published: April 2, 2012
© 2012 American Chemical Society
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(αS) in Parkinson’s disease.30−36 It is clear that these proteins
exert their neurotoxic effect through oligomerization and
fibrillization, but the chemical-scale details of the misfolding
process and the precise identity of the toxic species remain
unclear. Thioamides could be used to perturb hydrogen-
bonding interactions important to misfolding or as minimally
perturbing spectroscopic probes to monitor the misfolding
process itself. Kelly and co-workers have shown that some
amyloid proteins are tolerant of backbone substitution, making
oxoester and olefin isostere replacements of the amide bond to
study Aβ.37−39 The short (40−42 residue) length of Aβ makes
it a viable target for direct peptide synthesis, but other amyloid
proteins are too large to be accessed by direct solid-phase
peptide synthesis (SPPS). Brik, Lashuel, and co-workers have
shown that ubiquitin-modified αS can be made through native
chemical ligation (NCL), the most common and robust
segment condensation reaction for unprotected peptides.40,41
We sought to build off of their work in order to use αS as a test
case for the incorporation of backbone thioamides into full-
sized proteins. If successful, this would provide us with labeled
αS for folding studies and test the general compatibility of
thioamides with NCL for protein semi-synthesis. Fluorescence
quenching studies with thioamidous αS can be used to make
models of structural changes during aggregation.
Scheme 1. (Top) Native Chemical Ligation Mechanism and
(Bottom) Potential Thioamide Side Reactions
In spite of their potential utility, the synthesis of thioamide-
containing proteins has been limited to single chains assembled
on solid phase. The longest fully synthetic thiopeptides are the
35 amino acid leucine zippers reported by Miwa and co-
workers and the Villin headpiece variant HP35 described in
Goldberg et al.14,26 Fischer and co-workers were able to
reconstitute a functional ribonuclease S (RNase S) through the
noncovalent association of a proteolytic fragment of RNase S
and a synthetic thiopeptide.8,42 However, the methods
employed with RNase S clearly cannot be used for the general
construction of thioamide-containing proteins. There have
been, to our knowledge, no systematic studies of the
compatibility of thioamides with NCL reactions.
NCL reactions require that one peptide bear an N-terminal
Cys (or Cys surrogate) and that the other peptide be activated
as a C-terminal thioester. The two fragments initially engage via
reversible transthioesterification, followed by an irreversible S-
to-N acyl shift to generate a “native” amide bond (Scheme 1,
top). NCL reactions have been shown to be tolerant of protein
functional groups as well as a variety of non-native functional
groups.43 Thus, we expected thioamides to be compatible with
NCL conditions, although we had some concerns regarding
Edman-type side reactions or Cys desulfurization through
attack of the nucleophilic thioamide (Scheme 1, bottom).
Another potential concern was desulfurization of the thioamide
via transient water attack at the thiocarbonyl carbon followed
by expulsion of H2S from the tetrahedral intermediate.44,45
We began by synthesizing thioamide-containing C-terminal
thioesters or N-terminal Cys peptides and testing their
compatibility with NCL. Since the HF cleavage step of Boc-
based peptide synthesis degrades thioamides, we used an Fmoc-
based method. We also tested several tools that would allow us
to make larger proteins: protecting groups for multiple
ligations, in situ thioester formation from masked precursors,
and finally ligation to expressed proteins. We applied the
considerations from these methodological experiments to the
successful synthesis of a thioamide-labeled α-synuclein, which
can be used in folding experiments using our thioamide
fluorescence quenching methods. [Note: Thioamide residues
are represented by the one or three letter codes of the
equivalent oxoamide amino acids with a prime symbol (e.g., L′
or Leu′ represent thioleucine).]
RESULTS AND DISCUSSION
■
To examine the stability of thioamides toward NCL reaction
conditions, our initial tests used thioesters formed by solution-
phase thioesterification for ease of synthesis. We have
previously shown that Fmoc-protected thiocarboxybenzotria-
zoles can be used with conventional Fmoc-protected amino
acids in the solid-phase synthesis of thioamide-containing
peptides. These syntheses are typically carried out on 2-
chlorotrityl resin so that deprotection and cleavage can be
carried out with moderate (60−80%) TFA concentrations. This
method required no modification for the synthesis of the N-
terminal Cys fragments and only minor modification to
generate C-terminal thioesters. For a thioester, the peptide
was synthesized and cleaved with an 8:1:1 CH2Cl2/AcOH/TFE
solution that afforded the peptide with its N-terminus and side-
chain protecting groups intact. The C-terminus was converted
to a thioester using PyBOP and thiophenol, after which the
acid-labile protecting groups were removed with TFA. The
products were HPLC purified to give thioamide-containing C-
terminal thioesters.
Solution-phase thioesterification of protected peptides can
lead to epimerization of the α-carbon of the C-terminal residue.
However, studies have shown that a judicious choice of
activating agent and short reaction times can reduce
epimerization.46,47 We find that using PyBOP and thiophenol
for reaction times of less than 1 h is effective. For example, the
activation of Ac-XVA (X = 7-methoxycoumarin-4-yl alanine)
was analyzed by HPLC through comparison to an authentic
sample of the epimer containing D-Ala, and the yield of
epimerized thioester was found to be 1−8% (see Supporting
Information). Since the focus of this study is on the reactivity of
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the thioamide, our initial test peptides featured a C-terminal
Gly to avoid concerns about stereochemical integrity.
We generated a series of peptides with strategically placed
thioamides and explored their reactivity under standard NCL
conditions with either DTT or TCEP as the reducing agent.
Reactions were monitored by HPLC (quantified by LC area
percentage, LCAP) and MALDI MS or MS/MS. The trial
sequences were selected to determine the NCL compatibility of
thioamides placed in either the thioester (Table 1, C) or Cys-
Table 1. Thioamide Peptide Test Ligations
a
b
b
peptides
Red.
conversion
purity
A.
B.
C.
D.
Ac-AKXAGCOSPh (1)
+ CL′AKWAA (2)
DTT
86% (18 h)
77% (19 h)
99% (19 h)
62% (4 h)
63% (19 h)
55% (3 h)
n/a
74% (4 h)
71% (4 h)
81% (4 h)
54% (4 h)
83% (8 h)
54% (3 h)
n/a
TCEP
DTT
Ac-AKXAGCOSPh (1)
+ CAGL′KXAG (3)
Ac-GL′KXAGCOSPh (4)
+ CAGLKXAG (5)
TCEP
DTT
TCEP
Ac-GAKXL′GCOSPh (6)
+ CAGLKXAG (5)
a
Reactions carried out with 1 mM thioester peptide and 1 mM Cys-
Figure 1. HPLC analysis of ligation to form peptide C. The top three
panels show aliquots taken at different time points from a reaction
using DTT. The bottom panel shows an aliquot taken from a reaction
under otherwise identical conditions using TCEP as reductant.
Absorbances recorded at 325 nm.
peptide in phosphate buffer, pH 7, with 20 mM reductant (Red.)
under Ar. Conversion and purity determined at indicated times as
described in Supporting Information.
b
peptide (Table 1, B) fragment. We also wished to search for
possible side reactions of thioamides directly adjacent to the
thioester (Table 1, D) or Cys residue (Table 1, A). The peptide
sequences are given in Table 1. Peptides A−C were synthesized
successfully, but peptide D could not be synthesized because
the thioester could not be prepared in pure form. For reaction
D, although epimerization could be avoided, the Edman-type
cyclization shown in Scheme 1 and subsequent side reactions
prohibited the preparation of the thioester 6. In order to
understand these side reactions, we subjected Boc-Ala′Gly to
our thioesterification conditions. MS and NMR characterization
of the intermediates was consistent with phosphonium-
mediated deoxygenation followed by reaction with thiophenol
(see Supporting Information).
NCL reactions among the test peptides shown in Table 1
were carried out at 1 mM concentrations in Ar-sparged pH 7
phosphate buffer in the presence of 20 mM DTT or TCEP. For
thiophenyl esters, no additional thiophenol was used. Reactions
were monitored by HPLC; product and intermediate peaks
were identified by MALDI MS. Peak assignments were
confirmed by comparison to authentic samples synthesized
directly on solid phase. In order to ascertain that no
desulfurization occurred, oxoamide versions of some peptides
were also prepared and their HPLC retention times compared
to ligation HPLC traces. HPLC analysis of peptide C synthesis
is shown in Figure 1; others are shown in Supporting
Information.
ligated products was achieved at different time points than
maximal purity, and they are therefore listed separately in Table
1.
When the preformed disulfide-bonded thiopeptide dimer
(L′AF*LCKAXG)2 or its oxoamide equivalent (LAF*LCK-
AXG)2 was incubated with TCEP, side products unique to the
thioamide sequence were observed. Cysteine-free thiopeptide
L′AF*LAKAXG and oxopeptide monomer LAF*LAKAXG
were unaffected by overnight incubation in TCEP buffer. Since
no significant side products are observed in DTT ligations, and
the TCEP-based side reactions occur only when disulfides are
present, we hypothesize that they are initiated by attack of the
thioamide sulfur on the thiophosphonium byproduct of
disulfide cleavage.48−50 HPLC analyses of these TCEP
reactions and our mechanistic hypothesis are included in
Supporting Information. Importantly for synthetic consider-
ations, these side reactions can be avoided by limiting initial
disulfide bond formation through careful Ar sparging.
While these initial reactions established the fundamental
compatibility of thioamides with NCL, several aspects needed
further development in order to incorporate thioamides into
proteins such as αS. First, we were still limited in size. A single
NCL reaction can, at best, produce an 80mer product from two
40mers (lengths are more limited for Fmoc-based SPPS;
70mers have been reported for NCL from Boc-based
SPPS).40,51 Second, the manner in which we originally formed
thioesters was limited to C-terminal Gly or low thioester-
ification yields of other amino acids (from short reaction times
to prevent epimerization). Third, we had not tested ligation
under “protein” conditions, especially in the presence of
denaturants. Finally, to access full-sized proteins, it is ideal if
one can ligate the synthetic peptide to an expressed protein
fragment in order to minimize synthesis of unmodified portions
of the protein. To expand the potential application of
thioamides through NCL, we began by testing conditions for
Reactions of these short peptides were typically rapid, with
greater than 50% conversion observed after only a few minutes.
The rate of conversion of the remaining thioester starting
material depended on the addition of excess thiophenol or 4-
mercaptophenylacetic acid (MPAA). Similar results were
observed at early time points when reactions were carried out
in 20 mM TCEP. However, overnight incubation with TCEP
resulted in a number of degradation products unless a
rigorously oxygen-free atmosphere was maintained (Figure 1,
bottom). In some cases, maximal yield (conversion) of the
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Figure 2. Multiple ligations using Thz (CZ) protection of N-terminal Cys. (Left) Reaction scheme with exact masses of reactants and products.
Sequences: 7, CZKKLAQXGG-COSR; 2, H2N-CL′AKWAA-CO2H; 9, H2N-CKKLAQXGGCL′AKWAA-CO2H; 10, Ac-AKXAG-COSR; 11, Ac-
AKXAGCKKLAQXGGCL′AKWAA-CO2H. (Right) HPLC chromatograms and MALDI mass spectra of indicated products. * indicates
contaminant. Yields are based on peak integration of HPLC traces shown in Supporting Information.
multiple ligations using N-terminal thiazolidine (Thz, Cz)
protection in one peptide fragment.
and MacMillan, uses a peptide connected to a Cys-Pro-Gly/Gla
linker, where Gla (Go) is glycolic acid.62,63 Our method uses a
Csb-Pro-Gla linker, where Csb (Cb) is a t-Bu-protected Cys;
the linker as a whole is hereafter referred to as CbPGo. We
focused on the CbPGo linker because of the ease of synthesis
(protected Cys hydroxy acid synthesis requires five steps).60
We used a slightly different method than the published route,
beginning by attaching bromoacetic acid to Rink amide resin.
Pro and then a dipeptide of the C-terminal amino acid and Csb
are attached, and the rest of the peptide is elongated by
standard Fmoc-peptide synthesis. After acidic cleavage from the
resin, the Csb t-Bu group (inert to TFA) is removed by
treatment with TCEP, initiating a cascade of reactions resulting
in a peptide thioester attached to the diketopiperazine (Dkp)
product of CbPGo rearrangement (Scheme 2). The Dkp
thioester can then either react directly with an N-terminal Cys
or undergo transesterification with a thiol additive like MPAA.
We first tested this strategy by synthesizing thioester 4 from
Table 1 (Ac-GL′KXAG-COSR) with the CbPGo linker. The
rearrangement, transthioesterification, and ligation can be
carried out in one pot, where a peptide such as Ac-
GL′KXAGCbPGo (12a) is added to a solution with TCEP
and a ligation partner, in this case Cys (Figure 3). TCEP
reduction of the disulfide is rapid and efficient (Supporting
Information). Cyclization is difficult to monitor directly, since
the Dkp thioester exchanges readily and is rapidly converted to
ligated product. If the reaction is carried out at a low
concentration of Ac-GL′KXAGCbPGo in the absence of Cys,
intermediates can be observed, such as the Dkp thioester, the
branched Ac-GL′KXAG thioester dimer, and the hydrolyzed
Ac-GL′KXAG carboxylic acid (Supporting Information).
Kent and others have shown that protection as Thz can be
used to block an N-terminal Cys that can then be revealed by
treatment with methoxylamine hydrochloride.52,53 We wanted
to ensure that MeONH2·HCl incubation would not degrade
the thioamide. Therefore, we synthesized three peptide
fragments (Figure 2: 7, CZKKLAQXGG-COSR; 2, H2N-
CL′AKWAA-CO2H; and 10, Ac-AKXAG-COSR). First,
peptides 7 and 2 were ligated under standard conditions to
form 8(Thz) (Figure 2, top right), and then the product was
deprotected with MeONH2·HCl to give peptide 9(Cys)Red.
.
HPLC and MALDI analysis showed that the expected product
was formed without significant byproducts. Although intra-
molecular disulfide-bonded 9(Cys)Ox macrocycles formed upon
standing, these could be reduced by TCEP for subsequent
ligations (Figure 2, middle right). Peptide 9 was then reacted
with a large excess of peptide 10. The product, peptide 11, was
confirmed by MADLI MS (Figure 2, bottom right). This set of
experiments showed that thioamides are inert toward methoxyl-
amine treatment and confirmed the functional group
compatibility observed in the test reactions.
Thioesterification by activation with PyBOP was useful for
the initial test reactions, but the possibility of epimerization and
the difficulty of solubilizing fully protected peptides for C-
terminal activation make this an undesirable long-term solution.
Therefore, we examined on-resin thioesterification methods.
“Safety-catch” linkers and related hindered amides require
alkylation of the sulfonamide/amide nitrogen by electrophiles,
which could react with the thioamide sulfur.54−57 On the other
hand, thioesters masked as oxoesters or even as amides can be
synthesized on solid phase, and then rearrangement to the
thioester can be initiated by reduction of a disulfide. Two
methods using masked thioesters have been published in the
literature. One strategy, reported by Muir, Botti, and others,
uses a peptide connected as an oxoester to a t-Bu-protected Cys
hydroxy acid.58−61 Another strategy, described by Kawakami
To demonstrate the compatibility of thioamides with NCL
conditions typical of full-sized proteins, we synthesized the
Villin headpiece HP35 proteina small 35 amino acid protein
that we have previously studied using thioamide FRET (Figure
4).14 Although 35mers are synthetically accessible without
ligation, their convergent synthesis from two fragments would
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Scheme 2. Thioester Generation by N-to-S Rearrangement
Figure 4. HPLC analysis of Villin HP35 synthesis by NCL. VillinN
thiophenyl ester synthesized by PyBOP activation. Aliquots taken at
different time points from a reaction using TCEP as reductant.
Absorbance recorded at 215 nm.
allow us to prepare versions with thioamides at different
locations in a combinatorial fashion. More importantly, ligation
for proteins of this size allows us to use protein ligation
conditions, but the Villin product is small enough that we can
monitor desulfurization by HPLC retention and MALDI MS.
Here, we ligated a 17mer thioester fragment and a 18mer Cys-
peptide. The thioester fragment was synthesized by activation
of the protected peptide with PyBOP and thiophenol in DMF.
Thioesterified peptide was recovered by precipitation through
addition of water and deprotected with TFA for ligation.
Ligation reactions, carried out in phosphate buffer with 20 mM
TCEP, saturating thiophenol, and 6 M Gdn·HCl, reached
completion with respect to the limiting VillinN peptide in about
4 h, with no TCEP side reactions or desulfurization observed.
This laid the groundwork for using NCL reactions to attach
thioamides to expressed protein fragments.
In order to make labeled αS as a first test of thioamide
incorporation into proteins through semi-synthesis, we
generated constructs for producing a version of αS with an
N-terminal His tag which could be removed by proteolysis to
reveal an N-terminal Cys for ligation. We chose to use
commercially available Factor Xa protease, which cleaves an
IEGR sequence and had been used previously in expressed
protein ligation.1,64 Since αS has no native Cys, we introduced
one by altering the expression construct to remove the first
eight residues and leave an N-terminal Cys (αS9−140C9) after
proteolysis. We chose position 9 because Trexler et al. had
previously studied αS with a Ser-to-Cys mutation at this
position in FRET experiments.65 The scheme for the semi-
synthesis of Val3-labeled αS is shown in Figure 5.
The C-terminal fragment, HTag-αS9−140C9 (HTag-14), was
expressed in E. coli, purified by Ni-NTA chromatography, and
proteolyzed with Factor Xa to give αS9−140C9 (14). PAGE gel
analysis of these steps (Figure 5, top right) shows that
αS9−140C9 was pure and fully cleaved at this point. Additional
cleanup could be carried out by HPLC purification using a C4
reverse phase column as necessary. The N-terminal fragment
was synthesized on solid phase using the CbPGo linker, with a
thioamide inserted at Val3 during SPPS (αS1−8V′3-CbPGo, 15).
Activation of the peptide by TCEP yielded αS1−8V′3 Dkp
thioester, which reacted with αS9−140C9 in situ to give full-length
αSV′3C9 (16). Although ligations reached only 50% com-
pletion, ligated αSV′3C9 could be purified by HPLC for analysis
Figure 3. HPLC and MALDI MS analysis of thioester formation from
Csb-Pro-Gla (CbPGo) linker. Cleaved, purified thioester precursor Ac-
GL′KXAGCbPGo-NH2 (12a) was incubated with TCEP and Cys at
pH 8.4. (Top) Representative HPLC chromatogram at t = 120 min
showing deprotected reactant peptide Ac-GL′KXAGCPGo-NH2 (12,
red circles), Cys-ligated product Ac-GL′KXAGC-CO2H (13, blue
squares), and side products (green triangles). Absorbance recorded at
325 nm. Inset: MALDI MS of product HPLC peak (23.7 min
retention time, m/z calcd 873.3). (Bottom) Relative concentrations of
reactant, product, and side products determined from HPLC
chromatograms. Labels are as above. Reactant decay and product
growth (sum of product and side products, orange diamonds) fit to
single exponential functions to obtain half-lives. See Supporting
Information for HPLC data, MS characterization, and fitting
procedures.
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Figure 5. Synthesis of labeled αS by ligation of a thioamide peptide to an expressed protein fragment. (Left) C-Terminal fragment of αS9−140 with an
N-terminal His tag, an S9C mutation, and IEGR cleavage sequence (HTag-14) expressed and purified on Ni-NTA resin. After Factor Xa cleavage to
generate an N-terminal Cys (αS9−140C9, 14), the protein fragment is ligated to synthetic αS1−8 with a thioamide at position 3 (αS1−8V′3), generated in
situ from precursor αS1−8V′3-CbPGo (15). (Top right) PAGE gel analysis of ligation. (Far right) MALDI MS of full-length αSV′3C9 (16, calcd m/z
14 536) or trypsinized αSV′3C9, giving αS1−6V′3 (17, calcd m/z 828.4). There is no peak corresponding to desulfurized oxopeptide αS1−6 (calcd m/z
812.4).
(see Supporting Information). The integrity of the thioamide
bond after ligation was assessed by MALDI MS of the full-
length protein and trypsinized products, as well as by UV
absorption spectroscopy. Edman-type degradation from thio-
carbonyl attack at the F4/M5 amide would result in a loss of the
four N-terminal residues, amounting to 552.2 Da. This was not
observed in MALDI MS of the ligated αSV′3C9 (Figure 5).
However, this does not address sulfur exchange with oxygen or
some more subtle modification. Trypsin digestion releases a
peptide corresponding to residues 1−5 with a mass of 828.4 Da
for the V′3 thiopeptide and 812.4 Da for the oxoamide peptide.
MALDI MS of trypsinized αS showed only the mass of the
intact thiopeptide, αS1−6V′3 (17) (Figure 5). Finally, compar-
ison of the UV absorption spectra of αSV′3C9 and αS9−140C9
indicates that 99% of the thioamide is intact after ligation
(Supporting Information). By all of these measures, the
thioamide appears to have been incorporated without
degradation.
Finally, in order to demonstrate the value of incorporating
thioamide probes into a protein such as αS, we carried out an
aggregation experiment in which intramolecular conformational
changes were monitored in an αS construct. αS, like other
amyloidogenic proteins, is known to aggregate first into soluble
oligomers of two to hundereds of subunits, and then into
longer fibrils which can tangle to form Lewy bodies.30,31,68,69
Since there is a great deal of uncertainty as to the structures of
the oligomers and the structural changes that lead to
fibrillization, a method for site-selectively monitoring conforma-
tional change could be very valuable to understanding αS
pathology. Indeed, many fluorescence-based studies have
attempted to determine structures for aggregation intermedi-
ates, but they often employ fluorophores that can perturb the
structure, particularly when substituted at sites that do not
natively contain an aromatic amino acid.70−81 Our thioamide
probe, which has been shown to quench Trp, Tyr, and the
unnatural amino acid Cnf, should offer much greater freedom,
particularly in the placement of the acceptor (i.e., the
thioamide) in a donor/acceptor pair and can be used to access
the stretch from residues 40 to 93, which contains no aromatic
amino acids.
We employ an αS construct with a single Trp donor
fluorophore (W94) and a single thioamide quencher (V′3). As
noted above, our previous work has shown that Trp
fluorescence is quenched by thioamides in a distant-dependent
manner.15 The native αS sequence has no Trp residues, so
selective excitation of an introduced Trp can be achieved with
295 nm light. Comparison to an oxoamide control (i.e., αSW94)
allows us to isolate thioamide quenching effects from other
environmental effects on Trp fluorescence.82 Since through-
space quenching does not occur over distances longer than 30
Å, observation of Trp fluorescence quenching normalized to
oxoamide controls shows that the Trp must be within 30 Å of
the thioamide residue.15 However, in an oligomer or fibril,
quenching can arise through either inter- or intramolecular
interactions. In order to isolate intramolecular misfolding
events, we carry out aggregation experiments with our
αSV′3C9W94 construct present in a 1:30 ratio with WT αS. A
ratio of less than one Trp/thioamide-labeled αS per 26 WT αS
should ensure that, on average, no two labeled αS are directly
next to each other in aggregates. This derives from a simple
statistical packing model in which each monomer unit would be
at the center of a cube with 26 partners along the centers of
faces and vertices, or at corners.
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The construct used in aggregation studies (18) was
synthesized by ligation of the same synthetic fragment
(αS1−8V′3) with an expressed C-terminal fragment
(αS9−140C9W94). For an aggregation experiment, the presence
of non-native Cys in the sequence could be problematic as the
formation of disulfide-bonded dimers would alter the
aggregation mechanism.66,67 Therefore, we incubated αS with
β-mercaptoethanol (BME) to prevent disulfide bond formation.
No significant increase in aggregation rate was observed in the
Cys mutants.
Aggregation experiments were carried out by shaking the
1:30 αSV′3C9W94/αS mixtures at 37 °C in phosphate-buffered
saline with BME. Although overall Trp fluorescence increased
over 4 days during protein aggregation, the relative fluorescence
of the thioamide (FThio/FOxo) decreased. (See Figure 6 for
αSW94/αS) in order to account for other local effects on
fluorescence, which are necessarily complex in an aggregate. We
monitored overall fibrillization by ThT fluorescence, as well as
the independent metrics of Congo Red staining and
sedimentation PAGE gel analysis (Supporting Information).
Comparing the ThT data to the Trp fluorescence data clearly
indicates that we can observe quenching in oligomers, which
occur at early time points (<24 h), before fibrillization and ThT
fluorescence (Figure 6). It is unlikely that multiple labeled αS
molecules are present in oligomers of less than 30 monomer
units. Therefore, quenching of Trp fluorescence provides
evidence of close intramolecular approach of residues 3 and
94 when bound in oligomers, consistent with some previous
studies showing folding of the N- and C-termini before
fibrillization.81 Our experiments show that thioamide fluo-
rescence quenching should be useful in monitoring inter-
mediates in the aggregation process, which are silent in the
conventional ThT protocol. Conducting many such experi-
ments in conjunction with positional scanning of the thioamide
probe should provide mechanistic insight into the aggregation
process and the mode of toxicity of these metastable
intermediates.
CONCLUSIONS
■
Thioamides can be valuable biophysical tools for perturbing
backbone hydrogen bonds, tracking protein motions, or
inducing cis/trans isomerization. Previously, their uses have
been restricted to peptides that can be synthesized on solid
phase. Here, we have shown that the thioamide functional
group is compatible with native chemical ligation conditions.
Furthermore, we have seen that thioamides are compatible with
thiazolidine protection of an N-terminal Cys, so that multiple
ligations can be used to construct larger proteins. To enable
facile syntheses of the requisite thioesters using Fmoc
chemistry, we adapted methods for the generation of a masked
thioester using the CbPGo linker. Deprotection of the t-Bu side-
chain protecting group can be carried out in situ so that the
thioester is generated and reacts rapidly with an N-terminal
Cys. Finally, we have shown that thioamidous peptides
synthesized with the CbPGo linker can be coupled to expressed
protein fragments by synthesizing a labeled version of αS.
We have recently shown that thioamide quenching of the
unnatural amino acid Cnf can be used to monitor protein
folding.14 In addition, thioamide quenching of Trp or Tyr could
be used to monitor interactions for native proteins.15 Most
significantly, we have found that visible wavelength fluoro-
phores can be quenched by thioamides through electron
transfer, so that folding experiments could be carried out with
site-selective fluorophore excitation in the presence of bio-
logical chromophores.83 However, all of these experiments
would be limited to thiopeptides synthesized on solid phase
without the ability to ligate thioamide-containing peptide
fragments. Thus, the methods developed here will be used to
synthesize thioamide-labeled proteins for use in the study of
protein folding and protein−protein interactions. Misfolded or
aggregating proteins are particularly attractive as they are often
refractory to higher resolution structural techniques such as
crystallography or NMR. We envision generating a large
number of labeled αS constructs and using thioamide
fluorescence quenching to monitor conformational changes
during aggregation and misfolding. Our proof-of-principle
experiment indicates that these studies are feasible and should
reveal information that cannot be accessed with common
Figure 6. Monitoring intramolecular misfolding during αS aggregation
using thioamide quenching. (Left) Monomeric αSV′3C9W94 (18)
mixed in a 1:30 ratio with WT αS. Aggregation assay carried out by
shaking at 37 °C for several days. Samples were taken daily, and Trp
fluorescence was measured at 350 nm. ThT was added, and
fluorescence at 490 nm was measured after 2 min incubation. Trp
fluorescence quenching is observed in oligomers and fibrils. ThT
fluorescence is only observed for fibril-bound ThT. (Top right)
Schematic representation of αSV′3C9W94 construct showing Trp,
thiovaline, and Cys. (Bottom right) Normalized Trp (squares) and
ThT (circles) fluorescence data for four aggregation experiments with
αSV′3C9W94 (Thio) and control experiments with αSW94 (Oxo). Both
Trp-labeled proteins were used in 1:30 mixtures with WT αS. See
Supporting Information for examples of primary fluorescence data and
a table containing normalized fluorescence values used in generating
this plot.
FThio/FOxo data, and see Supporting Information for primary
fluorescence data.) This indicates that positions 3 and 94
approach each other to within 30 Å in the oligomeric and
fibrillar states, but that they are more separated in the
monomeric state. Since we used a 1:30 αSV′3C9W94/αS ratio,
we interpret the quenching as arising primarily from intra-
molecular interactions within the labeled molecule. We
normalize our data to an equivalent oxoamide protein (1:30
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analytical column (Kyoto, Japan) using gradient 2 (Table S2). HPLC
chromatograms for the ligations in Table 1 are given in Figure S2.
Ligation of Model Thiazolidine-Protected Cys Peptides.
CzKKLAQXGG-S(CH2)2CO2Me 7 (1 equiv, 0.2 μmol, 1 mM) and
CL′AKWAA 2 (1 equiv, 0.2 μmol, 1 mM) were incubated in 200 μL of
a freshly made, argon-sparged ligation buffer (6 M Gdn·HCl, 100 mM
Na2HPO4, 25 mM TCEP, pH 7.2). The reaction was initiated by the
addition of thiophenol (1% v/v, 2 μL). After 22 h of stirring, the
reaction was quenched with 0.1% TFA in water. The isolated product,
CzKKLAQXGGCL′AKWAA 8, was purified by HPLC on a Vydac 218
TP C18 semiprep column using gradient 2 and characterized by
MALDI MS (Tables S2 and S3). The ligated peptide was treated with
100 μL of a deprotection solution (6 M Gdn·HCl, 100 mM Na2HPO4,
200 mM MeONH2·HCl, pH 4) for 8 h. The conversion from Thz-
thiopeptide to Cys-thiopeptide 9 was confirmed by MALDI MS
(Figure 2). The deprotected Cys-thiopeptide was purified by HPLC
on a Vydac 218 TP C18 semiprep column using gradient 2 (Table S2).
For the second ligation, the Cys-thiopeptide 9 (1 equiv, 0.009 μmol,
0.2 mM) and Ac-AKXAG-SPh 10 (2.5 equiv, 0.022 μmol, 0.5 mM)
were dissolved in the ligation buffer (same as above). The reaction was
initiated by the addition of thiophenol (4% v/v, 2 μL). The reaction
was purged with argon for 15 min and stirred under an argon
atmosphere at room temperature for 8 h. To monitor the ligation
progress, aliquots (10 μL) of the reaction solution were taken
periodically, diluted to 800 μL, and analyzed by analytical HPLC on a
YMC-Pack Pro C18 analytical column using gradient 2 (Table S2).
The final ligated product was confirmed by MALDI MS (Table S3).
Synthesis of CbPGo Peptides. Ac-GL′KXAGCbPGo 12a and Ac-
MDV′3FMKGLCbPGo (αS1−8V′3-CbPGo, 15) were synthesized on
Rink amide resin, each at 100 μmol scale. A synthetic scheme is given
in Supporting Information. The resin was first deprotected with 20%
piperidine in DMF and rinsed with DMF. Bromoacetic acid (0.0695 g,
500 μmol, 5 equiv) was pre-activated with DCC (0.0516 g, 250 μmol,
2.5 equiv) for 30 min in dry DMF (6 mL), and then coupled to the
resin over 30 min. Subsequently, Fmoc-Pro-OH (0.1687 g, 500 μmol,
5 equiv) was dissolved in dry DMF (6 mL) with DIPEA (174.3 μL, 1
mmol, 10 equiv) and coupled to the resin for 30 min.
The next two residues were introduced as a dipeptide to avoid
unwanted Dkp formation. For Ac-GL′KXAGCbPGo as an initial test,
Fmoc-Gly-Cys(S-t-Bu)-OH was synthesized on 2-chlorotrityl resin at
125 μmol scale with the general SPPS protocol, and cleaved with
CH2Cl2/AcOH/TFE (8:1:1 v/v). The crude product (0.0537 g, 110
μmol, 1.1 equiv) was dissolved in DMF (6 mL), with PyBOP (0.0572
g, 110 μmol, 1.1 equiv) and DIPEA (174.3 μL, 1 mmol, 10 equiv), and
then coupled to the Pro residue for 1 h. For αS1−8V′3-CbPGo, an
improved procedure was adopted, where Fmoc-Leu-Cys(S-t-Bu)-OH
was synthesized at 2 mmol scale in solution phase (a detailed
experimental procedure is given in Supporting Information). The
purified dipeptide (0.1634 g, 300 μmol, 3 equiv) was dissolved in
DMF (6 mL), with PyBOP (0.1561 g, 300 μmol, 3 equiv) and DIPEA
(174.3 μL, 1 mmol, 10 equiv), and then coupled to the Pro residue for
90 min. A second coupling was carried out to maximize the coupling
efficiency. The rest of both peptides were elongated and acetylated
with the standard protocol.
methods such as ThT binding. Of course, our methods are
potentially applicable to a great number of protein dynamics
questions, and we will continue to explore thioamide
compatibility with NCL reactions for the synthesis and study
of these proteins.
EXPERIMENTAL PROCEDURES
■
General Information. Fmoc-L-4-cyanophenylalanine (Fmoc-Cnf-
OH) was purchased from Peptech (Burlington, MA). Boc-L-
thionoleucine-1-(6-nitro)benzotriazolide, Fmoc-Gln(Trt)-OH, Fmoc-
Asn(Trt)-OH, and Fmoc-β-(7-methoxy-coumarin-4-yl)-Ala-OH (de-
noted X) were purchased from Bachem (Torrance, CA) or EMD
Chemicals (Philadelphia, PA). Benzotriazol-1-yl-oxy-tris-pyrrolidino-
phosphonium hexafluorophosphate (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, 2-
chlorotrityl chloride 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-diisopropylethylamine (DIPEA), thiophenol, benzyl mercaptan,
trifluoroacetic acid (TFA), tris(2-carboxyethyl)phosphine hydro-
chloride (TCEP), and Boc-4-thiazolidinecarboxylic acid (Boc-Thz-
OH) were purchased from Sigma-Aldrich (St. Louis, MO). All
deuterated solvents were purchased from Cambridge Isotopes
Laboratories, Inc. (Andover, MA).
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). Matrix-assisted laser desorption/ionization (MALDI)
mass spectra were collected with a Bruker Ultraflex III MALDI-TOF-
TOF mass spectrometer (Billerica, MA). Electrospray ionization (ESI)
mass spectra were collected with a Waters LCT Premier XE liquid
chromatograph/mass spectrometer (Milford, MA). UV/vis absorbance
spectra were obtained with a Hewlett-Packard 8452A diode array
spectrophotometer (Agilent Technologies, Santa Clara, CA). Fluo-
rescence spectra were collected with a Varian Cary Eclipse
fluorescence spectrophotometer fitted with a Peltier multicell holder
(currently Agilent Technologies). Nuclear magnetic resonance
(NMR) spectra were obtained with either a Bruker DRX 500 MHz
or DMX 500 MHz instrument.
Peptide Synthesis. All peptides were synthesized using a manual,
Fmoc-based solid-phase procedure described previously.14 Thioamide
monomers were synthesized using the general procedure previously
described for thioleucine.14 Explicit procedures for the synthesis of
thiovaline are given in Supporting Information. Off-resin peptide
thioesterfications are also described in Supporting Information. The
thiazolidine group was introduced as a Boc-4-thiazolidine carboxylic
acid and followed the general SPPS procedure.
Ligation of Model Thioamide Peptides. The N-terminal
peptide thioester 1 or 4 (1 equiv, 0.5 μmol) and the C-terminal
peptide fragment 2, 3, or 5 (1 equiv, 0.5 μmol) were dissolved in a
freshly made, argon-sparged ligation buffer (100 mM Na2HPO4, 20
mM DTT or TCEP, pH 7.0−7.2). A typical ligation began with the
combination of the two peptide fragments in a 5 mL, septum-capped,
and argon-purged round-bottom flask. The final volume of the
reaction was 500 μL, giving a 1 mM final concentration of each
peptide. The reaction mixture was sparged with argon for 15 min and
stirred under an argon atmosphere at room temperature. To monitor
the ligation progress, aliquots (50 μL) were taken periodically at
regular intervals and diluted to 800 μL with 0.1% TFA in water. Each
sample was analyzed by analytical HPLC on a YMC-Pack Pro C18
For Ac-GL′KXAGCbPGo 12a, the product was cleaved from the
resin with two treatments of CH2Cl2/TFA (7:3 v/v, 6 mL) on a
rotisserie, each for 30 min. The resulting solutions were combined,
dried by rotatory evaporation, and purified by HPLC on a Vydac 218
TP C18 semiprep column using gradient 12 (Tables S1 and S2). For
αS1−8V′3-CbPGo 15, thioanisole (500 μL) and TIPS (250 μL) were
added to the resin at 0 °C, followed by TFA (5 mL). The system was
stirred at 0 °C for 20 min and then allowed to warm to room
temperature over 30 min. The resulting solution was concentrated by
rotatory evaporation, from which the crude product was precipitated
with cold diethyl ether (10 mL) and purified by HPLC on a Vydac 218
TP C18 analytical column using gradient 10 (Tables S1 and S2).
Ligation of Ac-GL′KXAGCbPGo with Cysteine. A 50 mM
cysteine stock in water was freshly prepared. Peptide Ac-
GL′KXAGCbPGo 12a (0.1 μmol, ε325 = 12 000 M−1 cm−1) was
dissolved in H2O/CH3CN (11:4 v/v, 31.2 μL), and mixed with the
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cysteine stock (20.8 μL).84 A 2 μL aliquot of the above solution was
taken and diluted with 0.1% TFA in H2O (798 μL) as a t0 HPLC
standard. Next, 50 μL of a freshly prepared ligation buffer stock (400
mM Na2HPO4, 100 mM TCEP, pH 8.4) was added to initiate the
reaction. The reaction was placed in an incubator at 37 °C, shaking at
250 rpm. At appropriate time points, a 4 μL sample of the reaction
mixture was taken, quenched with 0.1% TFA in H2O (796 μL), and
analyzed by analytical HPLC on a YMC-Pack Pro C18 analytical
column using gradient 11 (Tables S2 and S3).
Native Chemical Ligation of Villin. The VillinN thioester (1
equiv, 0.2 μmol, 1 mM) and the VillinC fragment (2 equiv, 0.4 μmol, 2
mM) were dissolved in a freshly made, argon-sparged ligation buffer (6
M Gdn·HCl, 100 mM Na2HPO4, 20 mM TCEP, pH 7.2) to give a
final volume of 200 μL. The ligation was initiated by the addition of
thiophenol (1% v/v, 2 μL) and was stirred under an argon atmosphere
at room temperature. Aliquots (20 μL) of the reaction solution were
taken periodically, diluted to 800 μL, and analyzed by analytical HPLC
on a YMC-Pack Pro C18 analytical column using gradient 6 (Table
S2). In this method, VillinC, VillinN thioester, and ligated Villin eluted
at 14.8, 25.1, and 24.1 min, respectively (Figure 4). Ligated Villin was
isolated, dried in vacuo, and characterized by UV/vis spectroscopy and
MALDI MS (Table S3, Figures S9 and S10).
μmol, 1.5 mM) and expressed protein fragment αS9−140C9 (1 equiv,
0.06 μmol, 1 mM) were dissolved in 60 μL of a freshly made, degassed
ligation buffer (6 M Gdn·HCl, 200 mM Na2HPO4, 20 mM TCEP, 1%
v/v thiophenol, and 1% v/v benzylmercaptan, pH 8.0). The reaction
solution was placed in an incubator at 37 °C, shaking at 250 rpm. An
aliquot (30 μL) was removed from the solution at reaction time of 8 h
and quenched with 0.1% TFA in water (470 μL). The quenched
aliquot was dialyzed against Milli-Q water and analyzed by HPLC on a
Vydac 214 TP C4 analytical column using gradient 12 (Table S2). The
rest of the ligation solution was quenched at reaction time of 25 h,
dialyzed, and analyzed by HPLC. The product fractions were pooled,
dried in vacuo, and characterized by MALDI MS (Table S3).
Ligation of αS9−140C9W94 was carried out in a nearly identical
fashion with the following changes: Protein buffer was exchanged
using an Amicon (Millipore) Ultra 0.5 mL of 10 kDa spin column
prior to setup of the ligation reaction, and 2% v/v thiophenol was
used.
Trypsin Digest Analysis of αSV′3C9. αSV′3C9 16 (20 μg) was
dissolved in 18 μL of freshly prepared 25 mM NH4HCO3 (pH 7.5)
and digested with 2 μL of sequencing-grade modified trypsin (0.1 μg/
μL). The digestion was allowed to proceed at 37 °C for 4 h. An aliquot
(1.0 μL) of the digest was taken and analyzed by MALDI MS (Figure
5).
Overexpression and Purification of Recombinant αS9−140C9
and αS9−140C9W94 Mutants. A plasmid encoding HTag-αS9−140C9
(pSB6159) or HTag-αS9−140C9W94 (pSB7021) was generated as
described in Supporting Information. This plasmid was transformed
into E. coli BL21-(DE3) cells grown on agar plates. A starter culture of
4 mL of LB media was inoculated with a single colony and grown at 37
°C in the presence of ampicillin (100 μg/mL) for approximately 6 h. A
secondary culture of 500 mL of LB was inoculated with 1 mL of the
starter culture and grown at 37 °C in the presence of ampicillin. The
cultures were induced with 1 mM isopropyl-β-D-thiogalactopyranoside
(IPTG) at OD600 = 0.7 and allowed to grow at 25 °C overnight. Cells
were pelleted at 6000 rpm using a GS3 rotor and Sorvall RC-5
centrifuge for 15 min at 4 °C. After the supernatant was discarded, the
pellet was resuspended in 10 mL of resuspension buffer (50 mM Tris,
150 mM NaCl, 50 μM ethylenediaminetetraacetic acid (EDTA), pH
8.0, protease inhibitor cocktail, 1 mM phenylmethanesulfonyl fluoride
(PMSF), and 10 units/mL DNase1 grade II). The cells were lysed by
sonication, and the lysate was centrifuged at 13 200 rpm for 15 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 Tris, 150 mM NaCl, 20 mM TCEP, pH 8.0),
then twice with 10 mL of Wash Buffer B (50 mM Tris, 150 mM NaCl,
20 mM TCEP, 5 mM imidazole, pH 8.0), and twice with 10 mL of
Wash Buffer C (50 mM Tris, 150 mM NaCl, 20 mM TCEP, 30 mM
imidazole, pH 8.0). The protein was eluted with six 2 mL portions of
elution buffer (50 mM Tris, 150 mM NaCl, 20 mM TCEP, 250 mM
imidazole, pH 8.0). The eluted fractions were combined and dialyzed
against cleavage buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH
8.0) overnight. The protein that was not immediately proteolyzed was
stored at −80 °C until further use.
αS Aggregation Assays. Aggregation experiments were per-
formed according to literature precedent.73,79 First, 350 μL samples for
aggregation assays were prepared (97 μM WT αS with 3 μM αSW94 or
3 μM αSV′3C9W94) in phosphate-buffered saline (pH 7.0) containing 1
mM β-mercaptoethanol. Aggregation was seeded by the addition of
approximately 10% (wt/wt) pre-formed WT αS fibrils. The samples
were incubated at 37 °C for 4−6 days with continuous shaking at 1100
rpm. Periodically, 40 μL aliquots were removed to monitor changes in
tryptophan fluorescence and ThT or Congo Red binding. Protocols
and examples of primary fluorescence or absorbance data are given in
Supporting Information.
ASSOCIATED CONTENT
■
S
* Supporting Information
Procedures for peptide synthesis, protein expression, thioester
activation, and ligation reactions; analytical HPLC traces and
MALDI MS data; schemes illustrating proposed side reaction
mechanisms; primary aggregtion data, including Trp fluo-
rescence, ThT fluorescence, Congo Red absorbance, and PAGE
gel analysis. 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 by emailing the corresponding author.
AUTHOR INFORMATION
■
Corresponding Author
ejpetersson@sas.upenn.edu
Notes
Purification of αS9−140C9 or αS9−140C9W94. Ni-NTA-purified
HTag-αS9−140C9 (1 mg/mL) was proteolyzed with Factor Xa protease
(20 units per 1 mg of HTag-αS9−140C9) for 14 h at 37 °C without
shaking. The completion of proteolysis was confirmed by MALDI MS.
The reaction was quenched by the addition of 1 mM PMSF and 5 mM
TCEP and incubated for 30 min at room temperature. Subsequently,
the protein solution was incubated with 1 mL of Ni-NTA resin for 1 h
at room temperature to remove the N-terminal fusion peptide. The
resin was loaded into a column, and the flow-through was collected.
The resin was washed with 3 × 2 mL of cleavage buffer, and the
resulting solutions were combined with the flow-through. The
combined solution was dialyzed against Milli-Q water and further
purified by HPLC on a Vydac 214 TP C4 preparative column using
gradient 8 (Table S2). Purification of αS9−140C9W94 followed an
identical procedure.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by funding from the University of
Pennsylvania, Searle Scholars Program (10-SSP-214 to E.J.P.)
and the National Science Foundation (NSF CHE-1020205 to
E.J.P.). Instruments supported by the National Science
Foundation and National Institutes of Health include HRMS
(NIH RR-023444) and MALDI-MS (NSF MRI-0820996). The
authors thank Elizabeth Rhoades for the gift of the pT7 plasmid
containing α-synuclein. We thank Virginia Lee, John
Trojanowski, and members of the Center for Neurodegener-
ative Disease Research for the gift of the pRK172 plasmid and
for helpful advice. We also thank Cheryl McCullough for
Native Chemical Ligation of αS1−8V′3-CbPGo and αS9−140C9 or
αS9−140C9W94. Masked thioester αS1−8V′3-CbPGo 15 (1.5 equiv, 0.09
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(36) Yu, L. P.; Edalji, R.; Harlan, J. E.; Holzman, T. F.; Lopez, A. P.;
Labkovsky, B.; Hillen, H.; Barghorn, S.; Ebert, U.; Richardson, P. L.;
Miesbauer, L.; Solomon, L.; Bartley, D.; Walter, K.; Johnson, R. W.;
Hajduk, P. J.; Olejniczak, E. T. Biochemistry 2009, 48, 1870−1877.
(37) Bieschke, J.; Siegel, S. J.; Fu, Y. W.; Kelly, J. W. Biochemistry
2008, 47, 50−59.
assistance with peptide synthesis. S.B. thanks John Warner for
guidance with molecular biology. Cover art by Yanxin J. Wang.
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