Solving the α-conotoxin folding problem: Efficient selenium-directed on-resin generation of more potent and stable nicotinic acetylcholine receptor antaqonists

Article abstract of DOI:10.1021/ja910602h

α-Conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for diverse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce α-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an amphiphilic resin, we were able to chemically direct all five structural subclasses of α-conotoxins exclusively into their native folds. X-ray analysis at 1.4 A resolution of α- selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the -conotoxin fold. The -selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and α3β4, α7, and α1β1δγ nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.

Full text of DOI:10.1021/ja910602h

Published on Web 02/17/2010
Solving the r-Conotoxin Folding Problem: Efficient
Selenium-Directed On-Resin Generation of More Potent and
Stable Nicotinic Acetylcholine Receptor Antagonists
Markus Muttenthaler,† Simon T. Nevin,‡ Anton A. Grishin,|,‡ Shyuan. T. Ngo,§
Peng T. Choy,§ Norelle L. Daly,† Shu-Hong Hu,† Christopher J. Armishaw,⊥,†
Ching-I. A. Wang,† Richard J. Lewis,† Jennifer L. Martin,† Peter G. Noakes,‡,§
David J. Craik,† David J. Adams,|,‡ and Paul F. Alewood*,†
Institute for Molecular Bioscience, DiVision of Chemistry and Structural Biology, The UniVersity
of Queensland, Brisbane, Queensland 4072, Australia; Queensland Brain Institute, The
UniVersity of Queensland, Brisbane, Queensland 4072, Australia; and School of Biomedical
Sciences, The UniVersity of Queensland, Brisbane, Queensland 4072, Australia
Received December 16, 2009; E-mail: p.alewood@imb.uq.edu.au
Abstract: R-Conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors
(nAChR) with high specificity for diverse subtypes. Here we report the use of selenocysteine in a supported
phase method to direct native folding and produce R-conotoxins efficiently with improved biophysical
properties. By replacing complementary cysteine pairs with selenocysteine pairs on an amphiphilic resin,
we were able to chemically direct all five structural subclasses of R-conotoxins exclusively into their native
folds. X-ray analysis at 1.4 Å resolution of R-selenoconotoxin PnIA confirmed the isosteric character of the
diselenide bond and the integrity of the R-conotoxin fold. The R-selenoconotoxins exhibited similar or
improved potency at rat diaphragm muscle and R3ꢀ4, R7, and R1ꢀ1δγ nAChRs expressed in Xenopus
oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the
development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight
the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.
having distinct pharmacological and biophysical properties.3,4
The therapeutic potential of nAChRs is broad, with individual
subtypes being implicated in Alzheimer’s and Parkinson’s
disease, Tourette’s syndrome, pain, myocardial infarction,
nicotinic addiction, schizophrenia, attention-deficit hyperactivity
disorder, autism, epilepsy, depression, and anxiety.2,3 Subtype
selectivity of ligands is essential to sharply delineate physi-
ological functions and to minimize side effects in therapeutic
treatment of such diseases.5
Although much effort has focused on small molecule
antagonist development, little success has been achieved so far
in producing viable clinical candidates.2,6 By contrast, recent
research into marine cone snail venoms has revealed a rich
source of competitive, highly potent and selective antagonists
for a broad range of ion channels, including the nAChRs.7,8
The cone snails have evolved a highly sophisticated cell
machinery over 50 million years that allows the production of
bioactive peptides called conotoxins in a combinatorial fashion
for prey capture and survival strategies.8 Evolutionarily selected,
the R-conotoxins have nanomolar activity at target receptors
1. Introduction
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated
cationic channels that mediate fast synaptic transmission within
the peripheral and central nervous system.1,2 The nAChR is a
functional pentamer in which the five transmembrane spanning
subunits are symmetrically arranged around the central pore.1
Channel opening is induced by the endogenous neurotransmitter
acetylcholine (ACh) or exogenous agonists such as nicotine.2
The neuromuscular nAChR is made up of combinations of four
different subunits R1, ꢀ1, γ (or ꢀ in the adult), and δ, with a
predicted (R1)2ꢀ1γδ stoichiometry. By contrast, neuronal
nAChRs comprise homomeric or heteromeric combinations of
ligand binding R subunits (R2-R10) and structural ꢀ subunits
(ꢀ2-ꢀ4).2,3 These combinatorial possibilities generate a wide
diversity of subtypes in different locations in the nervous system
† Institute for Molecular Bioscience, Division of Chemistry and Structural
Biology, The University of Queensland.
‡ Queensland Brain Institute, The University of Queensland.
§ School of Biomedical Sciences, The University of Queensland.
| Present Address: Health Innovations Research Institute, RMIT Uni-
versity, PO Box 71, Bundoora, Victoria 3083, Australia
⊥ Present Address: Torrey Pines Institute for Molecular Studies, 11350
SW Village Pkwy, Port St Lucie, FL 34987, USA
(4) Gotti, C.; Zoli, M.; Clementi, F. Trends Pharmacol. Sci. 2006, 27,
482–491.
(5) Bunnelle, W. H.; Dart, M. J.; Schrimpf, M. R. Curr. Top. Med. Chem.
(Sharjah, United Arab Emirates) 2004, 4, 299–334.
(6) Arneric, S. P.; Holladay, M.; Williams, M. Biochem. Pharmacol. 2007,
74, 1092–1101.
(1) Unwin, N. J. Mol. Biol. 2005, 346, 967–989.
(2) Romanelli, M. N.; Gratteri, P.; Guandalini, L.; Martini, E.; Bonaccini,
C.; Gualtieri, F. ChemMedChem 2007, 2, 746–767.
(7) Lewis, R. J.; Garcia, M. L. Nat. ReV. Drug DiscoV. 2003, 2, 790–
802.
(3) Gotti, C.; Clementi, F. Prog. Neurobiol. (Amsterdam, Neth.) 2004,
74, 363–396.
(8) Terlau, H.; Olivera, B. M. Physiol. ReV. 2004, 84, 41–68.
9
3514 J. AM. CHEM. SOC. 2010, 132, 3514–3522
10.1021/ja910602h  2010 American Chemical Society

Solving the R-Conotoxin Folding Problem
A R T I C L E S
Figure 1. (a) Standard R-conotoxin synthesis. Assembly on resin using Boc-SPPS, followed by HF cleavage and random oxidation in aqueous buffer
solutions, yields all three possible disulfide isomers. (b) Parallel directed-folding strategy using selenocysteine. Parallel chain assembly was performed using
the HF stable SCAL linker on an amphiphilic ChemMatrix resin. Upon assembly, the conotoxins on solid support are transferred into labeled resin-bags,
followed by HF deprotection and oxidation in aqueous 0.1M NH4HCO3 buffers. Regioselective folding is induced upon HF deprotection due to selective
formation of the diselenide over the disulfide bond. Depending on the synthetic strategy, the peptides can then be separated, cleaved and purified individually,
or be cleaved and purified simultaneously via a single RP-HPLC run.
and an enormously refined selectivity that can differentiate
between individual subtypes of nAChRs.8,9 These peptides
contain 12-19 amino acids and have well-defined three-
dimensional structures, predominantly due to the bracing of the
backbone by two disulfide bonds in a Cys 1-3 and Cys 2-4
connectivity (referred to as the native globular connectivity).
Two loops are formed with a short turn-helix-turn backbone
feature that comprises most of the functional groups responsible
for activity and potency.9 The numbers of residues encompassed
within these two loops (m/n) represent the basis for a further
division into several structural subfamilies as the loop size
pairings correlate with the nAChR subtype target selectivity.8
Hence the R-conotoxins are ideal probes for receptor studies,
and are considered promising therapeutic agents for the treat-
ment of pain and other neurological disorders.2,7 However, the
challenge of obtaining correctly folded peptides has ensured that
in-depth investigations of R-conotoxins remain a low-throughput
process, thus hindering a plethora of potential structure-activity
studies.10
The major challenge is outlined in Figure 1a, where three
distinct folds with different disulfide bond connectivities and
functions may be obtained from the same linear sequence under
oxidative conditions. Although the oxidative folding process
may be partly influenced to give a dominant isomer by adjusting
oxidation buffers via variation in organic cosolvents, shuffling
agents, solute concentration, temperature and time, this empirical
approach often fails once non-native structural modifications
are embedded in the synthetic design.10,11 Furthermore, valida-
tion of the correct isomer is time intensive, as it involves
structure and disulfide bond connectivity determination or
biological characterization, if the biological target is known.
The purification process also adds to the complexity due to the
similar nature of the isomers, significantly lowering the yields.
Given that the primary reason to select chemical approaches
over peptide expression is to introduce non-native modifications
for improved structure-function, fluorescent tagging, ligation
chemistry, cyclization or PEGylation, it is essential to have
efficient regioselective control over disulfide bond formation.
Currently, this is achieved in a laborious fashion via orthogonal
thiol-protecting groups,12 which have long workup times and
low yields. We describe here a methodology that takes
advantage of preferential diselenide bond formation over
disulfide bond formation to selectively generate R-conotoxins
in their native fold either in solution or in a high throughput
parallel methodology. This approach gave easy access to all
five classes of nicotinic R-conotoxin antagonists with enhanced
stability and potency.
Selenocysteine (Sec, U) is considered to be the most
conservative substitution for cysteine and its isosteric character
has been exploited in a range of bioactive peptides to either
elucidate structure activity relationships (SAR) or to improve
their stability in reducing environments.13 Despite the resem-
blance between the elements selenium and sulfur, the amino
acids Sec and Cys exhibit significantly distinct chemical
properties. While Sec and Cys have similar electronegativity
(EN(S) ) 2.58, EN(Se) ) 2.55), Sec is a stronger nucleophile
and a better leaving group than its sulfur counterpart.14
Furthermore, Sec exhibits a much higher acidity than Cys (pKa
(Sec) ) 5.24-5.63, pKa (Cys) ) 8.25).15–17 Comparative studies
on diselenide, selenylsulfide, and disulfide bonds in linear
unconstrained glutaredoxin fragments as well as in folded
glutaredoxin 3 showed a difference of 111-166 mV in redox
(12) Moroder, L.; Musiol, H. J.; Schaschke, N.; Chen, L.; Hargittai, B.;
Barany, G. In Houben-Weyl, Synthesis of Peptides and Peptidomi-
metics; Goodman, M., Felix, A., Moroder, L., Toniolo, C., Eds.; Georg
Thieme Verlag: Stuttgart, 2001; Vol. E22a, p 384-423.
(13) Muttenthaler, M.; Alewood Paul, F. J. Pept. Sci. 2008, 14, 1223–
1239.
(9) Nicke, A.; Wonnacott, S.; Lewis, R. J. Eur. J. Biochem. 2004, 271,
2305–2319.
(10) Bulaj, G.; Olivera, B. M. Antioxid. Redox. Signal. 2008, 10, 141–
156.
(11) Nielsen, J. S.; Buczek, P.; Bulaj, G. J. Pept. Sci. 2004, 10, 249–256.
(14) Moroder, L. J. Pept. Sci. 2005, 11, 187–214.
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A R T I C L E S
Muttenthaler et al.
Table 1. Summary of Sequence and Bioactivity Data for R-Conotoxinsa
a Taken from ref 43.b Taken from ref 40.c Taken from ref 21.a Shown are the amino acid sequence, the replacement of pairs of cysteine (red C) by
isosteric selenocysteine (blue U), the selectivity towards correctly folded material (globular isomer), and the IC50 values (mean ( SEM, n ) 6) for
inhibition of ACh-evoked currents mediated by (R1)2ꢀ1δγ, R6R3ꢀ2, R3ꢀ4, R9R10, and R7 nAChR subtypes expressed in Xenopus oocytes. n.d., not
determined; * C-terminal amide.
potential between the disulfide and the diselenide bond.18,19 This
difference, in combination with the higher nucleophilicity and
lower pKa of Sec, suggests that diselenide or selenylsulfide bond
formation is highly favored over disulfide bond formation as
was observed in earlier studies.20 The lower redox potential of
the diselenide bond should at the same time provide higher
stability in a reductive environment.21
in dehydroalanine and consequently piperidyl adducts.22 Boc-
L-Sec(MeBzl)-OH was synthesized following the protocol of
Armishaw et al..21 Chain assembly of all R-conotoxin sequences
proceeded smoothly using HBTU-mediated Boc in situ neu-
tralization SPPS23 on a 4-methylbenzhydrylamine resin. The
linear peptides were deprotected and cleaved by hydrofluoric
acid (HF) in a single step. Intriguingly, the Sec residues were
found to be fully oxidized directly upon deprotection via HF
(pH < 1) and it was not possible to isolate the fully reduced
species. Attempts to do so in the presence of an excess of
reducing agents such as TCEP, TBP, or NaBH4 led to significant
deselenation. Oxidation of the diselenide, dithiol-containing
R-conotoxin intermediates was performed in 0.1M NH4HCO3
at pH 8.4 and the solution was analyzed by RP-HPLC and
LCMS (see Supporting Information Figure S2). Additional
oxidation trials on the selenium analogs of AuIB, MI, and
[A10L]PnIA revealed that once the diselenide is formed,
disulfide bond formation occurred readily at pH 5 in contrast
to the native all Cys peptides, which remain as free thiols (see
Supporting Information Figure S3). In general, oxidations (pH
8.4, 25 °C) were complete within 10 min without any folding
enhancers and only a single purification step was necessary.
This makes Sec-directed oxidative folding far more efficient
than standard oxidation protocols or orthogonal protecting group
strategies.
2. Results
2.1. Directed Folding using SelenocysteinesIn Solution. To
investigate the potential of selenocysteine to direct oxidative
folding in a straightforward and efficient manner, well studied
R-conotoxins from all five existing functional classes with
differing nAChR selectivity (R3/5-MI, R4/3-ImI, R4/4-BuIA,
R4/6-AuIB, R4/7-Vc1.1, R4/7-[A10L]PnIA) were chosen and
complementary pairs of cysteine residues in each sequence were
replaced by selenocysteine (see Figure 1b and Table 1). The
R-conotoxins AuIB, Sec[1,3]-AuIB, Sec[2,4]-AuIB, MI, Sec[1,3]-
MI, Vc1.1, Sec[1,3]-Vc1.1, [A10L]PnIA, Sec[1,3]-[A10L]PnIA,
Sec[2,4]-[A10L]PnIA, ImI, Sec[1,3]-ImI, Sec[2,4]-ImI, were
assembled using Boc-SPPS incorporating selenocysteine via
Boc-L-Sec(MeBzl)-OH. This chemical approach was chosen
as reports in the literature suggest that Sec racemization may
occur using Fmoc chemistry strategies. Furthermore, Sec was
shown to have a high tendency to deselenate via ꢀ-elimination
during iterative piperidine Fmoc-deprotection steps that result
2.2. Directed Folding using SelenocysteinesOn Resin. With
regioselective folding established in solution we set out to
develop resin-supported chemistry to underpin high throughput
parallel processing of peptides and unify time-intensive tasks
such as side-chain deprotection, oxidation, cleavage, and
purification to give fast and efficient access to correctly folded
R-conotoxins (see Figure 1b). R-Conotoxins MI, Sec[2,4]-MI,
Vc1.1, Sec[1,3]-Vc1.1, BuIA, Sec[2,8]-BuIA, and [A10L]PnIA
were assembled using Boc-chemistry on an amphiphilic resin
(ChemMatrix) with a C-terminal HF stable safety-catch acid
(15) Huber, R. E.; Criddle, R. S. Arch. Biochem. Biophys. 1967, 122, 164–
173.
(16) Nygard, B. Ark. Kemi 1967, 27, 341–361.
(17) Arnold, A. P.; Tan, K. S.; Rabenstein, D. L. Inorg. Chem. 1986, 25,
2433–2437.
(18) Besse, D.; Siedler, F.; Diercks, T.; Kessler, H.; Moroder, L. Angew.
Chem., Int. Ed. Engl. 1997, 36, 883–885.
(19) Metanis, N.; Keinan, E.; Dawson, P. E. J. Am. Chem. Soc. 2006, 128,
16684–16691.
(20) Pegoraro, S.; Fiori, S.; Rudolph-Bo¨hner, S.; Watanabe, T. X.; Moroder,
L. J. Mol. Biol. 1998, 284, 779–792.
(22) Moroder, L.; Besse, D.; Musiol, H.-J.; Rudolph-Bo¨hner, S.; Sideler,
F. Biopolymers 1996, 40, 207–234.
(21) Armishaw, C. J.; Daly, N. L.; Nevin, S. T.; Adams, D. J.; Craik, D. J.;
Alewood, P. F. J. Biol. Chem. 2006, 281, 14136–14143.
(23) Schno¨lzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. H.
Int. J. Pept. Protein Res. 1992, 40, 180–193.
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A R T I C L E S
Figure 2. Functional assays of R-conotoxins. (a) Inhibition of nerve-evoked muscle contraction of rat diaphragm by MI and its selenium analog at concentrations
of (i) 150 nM and (ii) 300 nM. Error bars are mean ( SEM (* P ) 0.0123, n ) 3, 2 way ANOVA) (b) Inhibition of ACh-evoked currents mediated by
nAChR subtypes expressed in Xenopus oocytes by R-conotoxin (i) MI, (ii) [A10L]PnIA, (iii) AuIB and their Sec-analogs. Error bars are mean ( SEM (n
) 6). The concentration-response data are summarized in the Table (iv), where IC50 is the half-maximal inhibitory concentration and nH is the Hill coefficient.
labile (SCAL) linker (see Supporting Information Figure S1).24
Simultaneous side-chain deprotection by HF of the conotoxins
separately packaged in labeled solvent-permeable 74 µm mesh
polypropylene bags25 delivered the fully deprotected peptides
on-resin and was followed by oxidation in 0.1M NH4HCO3
buffer at pH 8.4. Similar to solution folding outcomes, different
ratios of the three possible disulfide bond isomers of the all-
Cys analogs could be accessed by changing the folding
conditions (see Supporting Information Table S2 online).26 Full
regioselective control over multiple isomer formation was only
achieved by incorporation of isosteric selenocysteine into the
synthetic scheme (see Table 1 and Supporting Information
Figure S4). Once the peptides folded on-resin, a chemoselective
reaction (reductive acidolysis by NH4I/TFA) activates the linker
and releases the correctly folded R-conotoxins from the solid
support (see Supporting Information Figure S1). Depending on
the peptide library strategy, cleavage and purification can either
be achieved individually (separated by the labeled teabags) or
simultaneously in an efficient “one-pot” approach. Both alterna-
tives were investigated and in both cases the correctly folded
peptides were isolated in high purity (see Supporting Information
Figure S5).
followed by 300 and 500 nM. Over a 2 h period, the globular
selenium-analog Sec[2,4]-MI was the only peptide capable of
90% inhibition of the ACh-induced muscular contraction at 150
nM (see Table 1 and Figure 2a). Both peptides were able to
completely block the muscle contraction at concentrations of
300 and 500 nM.
2.3.2. ElectrophysiologyAssaysusingXenopusOocytes.R-Cono-
toxins MI, [A10L]PnIA and AuIB are selective for the muscle
R1ꢀ1γδ and the neuronal R7 and R3ꢀ4 nAChR subtypes,
respectively, and the activity of the native peptides a well as
their selenium-analogs was examined on ACh-evoked currents
mediated by recombinant nAChRs expressed in Xenopus oocytes
(see Table 1 and Figure 2b). Globular R-conotoxin MI was
tested on ACh (1 µM)-evoked currents mediated by muscle
(R1)2ꢀ1γδ nAChR. Concentration-response curves obtained for
the inhibition of ACh-evoked currents by globular MI exhibited
an IC50 of 26 nM and a Hill coefficient of 1.1 (n ) 6). Globular
Sec[2,4]-MI retained the potency with an IC50 of 9 nM and Hill
coefficient of 0.9 (n ) 6). Globular [A10L]PnIA was tested on
ACh (100 µM) -evoked currents mediated by neuronal R7
nAChRs and exhibited an IC50 of 23.3 nM and a Hill coefficient
of 2.0 (n ) 6). Globular Sec[1,3]-[A10L]PnIA had a similar
IC50 value of 15.5 nM and a Hill coefficient of 1.3 (n ) 5).
Neuronal R3ꢀ4 nAChR-mediated currents evoked by 50 µM
ACh were inhibited by globular AuIB giving an IC50 value of
3.1 µM and a Hill coefficient of 1.5 (n ) 6). The selenium
substituted globular Sec[1,3]-AuIB and globular Sec[2,4]-AuIB
exhibited IC50’s of 0.3 µM and 1.1 µM and Hill coefficients of
2.0 and 1.0 respectively (n ) 6).
2.3. Biological Characterization. 2.3.1. Contraction Assay
Rat Diaphragm. The biological activities of globular MI and
Sec[2,4]-MI were characterized by their ability to inhibit
acetylcholine (ACh)-induced muscular contractions in response
to phrenic nerve stimulation in rat diaphragm preparations. The
peptides were initially tested at a concentration of 150 nM,
(24) Patek, M.; Lebl, M. Tetrahedron Lett. 1991, 32, 3891–3894.
(25) Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley,
C. T.; Cuervo, J. H. Nature (London) 1991, 354, 84–86.
2.4. Structural Characterization. The enhanced potencies of
the selenium analogs prompted a structural analysis of the
isosteric character of selenocysteine. Circular dichroism (CD)
(26) Brust, A.; Tickle, A. E. J. Pept. Sci. 2007, 13, 133–141.
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Muttenthaler et al.
Figure 3. Structural analysis of R-conotoxin analogs. a(i) CD analysis of the globular AuIB and its selenocysteine analogs showing the presence of an
R-helix. a(ii) Overlay of the RH NMR shifts of globular AuIB and its selenium analogs showing no significant changes in overall structure. b(i) Superimposition
of the crystal structures of R-PnIA and R-Sec[2,4]-[A10L]PnIA illustrating the isosteric character of the diselenide bond (red) and the disulfide bond (yellow).
b(ii) 2Fo-Fc electron density around the diselenide bond of Sec[2,4]-[A10L]PnIA, contoured at 1σ. b(iii) Physicochemical properties of sulfur and selenium
(black)14 and structural data (purple) of torsion angles, CR distances and bond lengths derived from the crystal structure analysis of Sec[2,4]-[A10L]PnIA.
analysis of globular and ribbon AuIB, and its selenium analogs
showed that the R helix was preserved in the native and in the
selenium analogs (see Figure 3a (i)). NMR analysis of RH
chemical shifts of peptides was conducted, as it is a rapid and
reliable way of detecting structural differences if present.27,28
It is clear from comparison of the RH chemical shifts of native
and selenocysteine isomers that there were no significant
differences in their three-dimensional structures (see Figure 3a
(ii)). X-ray analysis of Sec[2,4]-[A10L]PnIA and comparison
with the crystal structure of R-PnIA allowed high resolution
insight into bond lengths, CR distances and torsion angles (see
Figure 3b and Supporting Information Table S3). Sec[2,4]-
[A10L]PnIA was crystallized under similar conditions to those
described for PnIA,29 but the crystals grew isomorphously with
PnIB in the space group P212121. The selenocysteine mutant of
PnIA did not hinder the growth of well-diffraction crystals (1.42
Å resolution using in-house source). The refined structure of
Sec[2,4]-[A10L]PnIA comprises all 16 residues plus 27 solvent
molecules and has an overall Rfactor of 15.6% with Rfree of 17.1%.
Overall the structure of Sec[2,4]-[A10L]PnIA is very similar
to that of PnIA. The rms (Root Mean Square) deviation for
superimposition of main chain atoms between Sec[2,4]-
[A10L]PnIA and Pn1A is 0.4 Å. The conformation of the
diselenide bridge of selenocysteine mutant of PnIA is also
similar to that of the disulfide bridge of the native peptide. The
dihedral angles (Cꢀ-S-S-Cꢀ or Cꢀ-Se-Se-Cꢀ) of Sec[2,4]-
[A10L]PnIA are -98.3° and -93.9° for Cys1-Cys3 and Sec2-
Sec4, respectively, and compares well with -98.6° and -83.1°
for the disulfide counterparts in PnIA. The distance is 5.7 Å
for the diselenide bridge Sec2-Sec4 CR atoms and 5.1 Å for
Cys1-Cys3 CR atom in Sec[2,4]-[A10L]PnIA, with a bond
length of 2.33 Å and 2.03 Å for the diselenide and disulfide
bridge, respectively. Similar values were observed in PnIA (5.1
Å for Cys1-Cys3 CR; 5.4 Å for Cys2-Cys4 CR, S-S bond
lengths of 2.03 Å). Structural analysis of the selenocysteine
mutant of PnIA supports the notion that replacement of S for
Se atoms in peptides or proteins does not induce significant
distortion either in the diselenide bridge or in the overall fold.30
Interestingly, C18-RP-HPLC coelution analysis showed that the
R-conotoxin with the most pronounced activity increase Sec[1,3]-
AuIB was the only analog that did not coelute its native
counterpart, indicating that the more pronounced hydrophobicity
of the diselenide bond might contribute to the observed change
in activity.
2.5. Stability Assays. RP-HPLC and LC-MS studies on
AuIB and the R-selenoconotoxin Sec[1,3]-AuIB and Sec[2,4]-
AuIB were performed in a solution containing equimolar
glutathione and in rat plasma at physiological pH. In both
systems LC-MS analysis demonstrated that a single diselenide
bond was able to largely suppress scrambling (see Figure 4).
The observed selenylsulfide to diselenide bond ratio during the
glutathione assay correlated well with the oxidation profile
observed after full reduction and reoxidation, and after oxidative
(27) Wishart, D. S.; Bigam, C. G.; Holm, A.; Hodges, R. S.; Sykes, B. D.
J. Biomol. NMR 1995, 5, 67–81.
(28) Wishart, D. S.; Sykes, B. D.; Richards, F. M. Biochemistry 1992, 31,
1647–1651.
(29) Hu, S. H.; Gehrmann, J.; Guddat, L. W.; Alewood, P. F.; Craik, D. J.;
Martin, J. L. Structure 1996, 4, 417–423.
(30) Strub, M.-P.; Hoh, F.; Sanchez, J.-F.; Strub, J. M.; Bock, A.; Aumelas,
A.; Dumas, C. Structure 2003, 11, 1359–1367.
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A R T I C L E S
Figure 4. Stability studies on AuIB and its selenium analogs. (A) Proposed disulfide bond scrambling mechanism in a 2-disulfide bond system. (B) Analytical
RP-HPLC traces of AuIB and Sec[1,3]-AuIB in stoichiometric equimolar 0.25 mM glutathione solution at physiological pH 7.4 (PBS buffer) and 37 °C,
showing the structural change from the globular into the ribbon isomer. (C) Selenium analogs significantly suppress scrambling in rat plasma and glutathione
solution. The degradation of the selenoconotoxins is very similar to the native analogs. Error bars are mean ( SEM for n ) 3.
workup. Further analyses showed that the stability of the Sec
analogs in rat plasma was similar to their native analogs with
a half-life of 24 h.
orthogonal thiol-protecting group strategies, providing the basis
for the folding of more complex disulfide rich peptides
containing 3 or 4 disulfide bond bridges, a task that has not
been achieved as yet.
The development of resin-supported chemistry compatible
with the selenium-directed folding approach provides the basis
for high-throughput production of correctly folded R-conotoxins.
The parallel processing of peptide assembly, side-chain depro-
tection, oxidation, cleavage, and purification allows efficient
production of a large number of peptides (see Figure 1b). Similar
to in-solution folding, different ratios of the three possible
disulfide bond isomers of the all-Cys analogs can be accessed
through astute selection of folding conditions, a process that
can be helpful when access to all different isomers is desired
(see Supporting Information Table S2).26 However, the use of
selenocysteine induces regioselective control also on-resin,
providing fast access to correctly folded R-conotoxins. This
methodology avoids excessive use of solvents and reagents, and
has the potential to be widely used for the synthesis of disulfide-
rich peptides.
In contrast to earlier studies on the selenocystine-containing
analogs of endothelin20 or apamin36 whose disulfide bonds do
not form a hydrophobic core we were aware of the possibility
that incorporation of Se-Se bond(s) could perturb the structure
of the more tightly compacted R-conotoxins. Thus, the synthe-
sized R-selenoconotoxins were examined closely to determine
any impact on structure and function. Strikingly, pharmacologi-
cal analysis of the R-conotoxin MI and its selenocysteine analogs
to inhibit muscle contractions in response to phrenic nerve
stimulation in rat diaphragm preparations revealed a significant
increase in potency for the globular selenium-analog Sec[2,4]-
MI (see Table 1 and Figure 2a). Quantitative electrophysiology
studies on R-conotoxins MI, AuIB, [A10L]PnIA and their
selenocysteine analogs using ACh-evoked currents of muscle
((R1)2ꢀ1δγ) and neuronal nAChR subtypes (R3ꢀ4, R7, R9R10)
3. Discussion
Strategic replacement of pairs of cysteine residues by sele-
nocysteine residues in R-conotoxins has a decisive impact on
the oxidative folding profiles (see Table 1 and Supporting
Information Figure S2). When placed into the native (globular)
positions (1-3, or 2-4) nearly quantitative formation of the
desired isomer was observed independent of the loop frame-
works for all five known classes of R-conotoxins. Intriguingly,
the Sec residues were found to be fully oxidized directly upon
deprotection via HF (pH < 1), which was unexpected as the
reported pKa(Sec) ) 5.24-5.63 would indicate that selenols
should be stable in strong acidic media. This observation is,
however, consistent with the paucity of literature describing that
selenocysteine can be only found either in the form of a
diselenide bond or as a selenolate stabilized in a catalytic
triads.31-33 The facile oxidation of the Sec residues and the rapid
oxidation (<10 min) at pH 8 of the two remaining Cys residues
drastically reduces workup times and makes Sec-directed
oxidative folding a far more efficient method than orthogonal
protecting group strategies (see Supporting Information Figure
S3 online). More importantly, this approach makes experiments
to determine disulfide bond connectivity superfluous and simpli-
fies the purification process. Furthermore, this methodology is
highly complementary to native chemical ligation19,34,35 and
(31) Syed, R.; Wu, Z. P.; Hogle, J. M.; Hilvert, D. Biochemistry 1993, 32,
6157–6164.
(32) Luo, G.-m.; Ren, X.-j.; Liu, J.-q.; Mu, Y.; Shen, J.-c. Curr. Med. Chem
2003, 10, 1151–1183.
(33) Epp, O.; Ladenstein, R.; Wendel, A. Eur. J. Biochem. 1983, 133, 51–
69.
(34) Quaderer, R.; Sewing, A.; Hilvert, D. HelV. Chim. Acta 2001, 84,
1197–1206.
(35) Gieselman, M. D.; Xie, L.; van Der Donk, W. A. Org. Lett. 2001, 3,
(36) Pegoraro, S.; Fiori, S.; Cramer, J.; Rudolph-Bo¨hner, S.; Moroder, L.
1331–1334.
Protein Sci. 1999, 8, 1605–1613.
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A R T I C L E S
Muttenthaler et al.
Figure 5. Surface analysis and binding model (a) Surface hydrophobicity plot of the cocrystal structure of AChBP and R-conotoxin PnIA with the hydrophobic
residues colored in orange and the disulfide bonds in yellow. R,ꢀ,γ indicate the complementary hydrophobic interaction of PnIA with the binding pocket.
(b) Close up of R-conotoxin PnIA binding into the hydrophobic pocket, revealing that the first disulfide bond1–3 is flanked by two tyrosine residues (3.6-3.9
Å). (c) NMR surface analysis of R-conotoxin AuIB, showing that the1–3 disulfide bond is in close proximity to the conserved hydrophobic residues that are
important for binding. The surface exposure of the disulfide/diselenide bonds of PnIA, Sec[2,4]-[A10L]PnIA and AuIB are analyzed in the table, showing
that the surface exposure of the diselenide bond is twice as large as the disulfide bond counterpart. (d) Co-elution study of AuIB its selenium analogs.
Analytical C18-RP-HPLC traces of the coelution of (i) globular AuIB with globular Sec[2,4]-AuIB and (ii) globular Sec[2,4]-AuIB with globular Sec[1,3]-
AuIB, revealed that Sec[1,3]-AuIB is more hydrophobic than its analogs.
expressed in Xenopus oocytes (see Table 1 and Figure 2b)
confirmed observed results obtained using the hemidiaphragm
preparation, with selenium analogs Sec[2,4]-MI, Sec[1,3]-AuIB,
Sec[2,4]-AuIB, and Sec[1,3]-[A10L]PnIA exhibiting up to a 10-
fold increase in inhibition compared to their respective native
R-conotoxins.
With selenium having a slightly larger atomic radius (S 1.02
Å, Se 1.17 Å) and longer bond length than sulfur (Cꢀ-Sγ )
1.82 Å, Cꢀ-Seγ ) 1.95-1.99 Å)14 we assessed the isosteric
character of selenocysteine more rigorously to determine if
structural differences could explain the increased biological
activity. CD and RH chemical shift NMR analysis of the AuIB
analogs showed that the secondary structure was highly
preserved in the selenium analogs, and no significant differences
in their three-dimensional structures from the native peptides
were observed (see Figure 3a). This conclusion was further
supported by the crystal structure of the R-selenoconotoxin,
Sec[2,4]-[A10L]PnIA (resolution 1.42 Å), which was near
identical to the native R-conotoxin PnIA (rmsd of 0.4 Å for the
main-chain atoms).29 Further structural analysis of Sec[2,4]-
[A10L]PnIA indicated that torsion angles, bond lengths, CR-CR
distances and electron densities of the diselenide and the
homologous disulfide bridge from the native PnIA were very
similar and no distortion was observed in the overall fold (see
Figure 3b and Supporting Information Table S3). To determine
the role of increased hydrophobicity of the Se-Se bond in
binding with the nAChR, the cocrystal structure of PnIA and
the acetylcholine binding protein (AChBP), a structurally and
functionally homologous protein of nAChR was examined.37
The cocrystal structure suggests that R-conotoxin PnIA binds
to AChBP via complementary hydrophobic patches, with the
[1,3]-disulfide bond flanked by two tyrosine residues (3.6-3.9
Å) (see Figure 5 a and b). This hydrophobic binding motif
(YNCCEEIY) (see Figure 5b) is also highly conserved in the
R3 subunit (YSCCPEPY), the subunit important for Sec[1,3]-
AuIB recognition, which has the most pronounced enhancement
(37) Celie, P. H. N.; Kasheverov, I. E.; Mordvintsev, D. Y.; Hogg, R. C.;
van Nierop, P.; van Elk, R.; van Rossum-Fikkert, S. E.; Zhmak, M. N.;
Bertrand, D.; Tsetlin, V.; Sixma, T. K.; Smit, A. B. Nat. Struct. Mol.
Biol. 2005, 12, 582–588.
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Solving the R-Conotoxin Folding Problem
A R T I C L E S
74 µm mesh polypropylene bags with the dimension of 5 × 5 cm.
Side-chain deprotection was achieved by 10 mL HF treatment at 0
°C for 2 h with p-cresol as scavengers [9:1 (v/v) HF:scavenger].
HF was evaporated and the solid support was directly transferred
into TFA to maintain the swelling properties. The resins were
washed with DCM, DMF, and H2O before being placed into a 0.1
M NH4HCO3 solution (100 mg /10 mL) at pH 8.4. The final
cleavage was performed either by reductive acidolysis with NH4I/
TFA/DMS for 1 h at 0 °C and 1 h at room temperature.26 TFA
was then evaporated by nitrogen purging, the peptides were
precipitated and the scavengers removed with cold ethyl acetate.
The peptides were redissolved in H2O, 0.1% TFA and lyophilized.
After MS and analytical RP-HPLC analysis, the peptides were
purified by C18-RP-HPLC.
4.3. Contraction Bioassay Rat Diaphragm and Electrophys-
iologysXenopus Oocytes. RNA preparation, oocyte preparation,
and expression of nAChRs in Xenopus oocytes were performed as
previously described.40 The methods and experimental protocols
for the rat diaphragm contraction bioassay and the electrophysi-
ological assays using Xenopus oocytes can be found in the Sup-
porting Information.
4.4. Circular Dichroism (CD) Spectroscopy. CD spectroscopy
was performed on a Jasco J-810 spectropolarimeter. Spectra were
recorded at room temperature under nitrogen atmosphere. Peptides
were dissolved in 20 mM phosphate buffer, containing 30%
trifluoroethanol at pH 7. The peptide concentration was determined
by quantitative RP-HPLC. The peptides were transferred into a
0.01 cm path length demountable cell and data were recorded over
5 scans, from 260 to 185 at 10 nm/min, with a resolution of 1 nm
and a response time of 0.25 s. CD data in ellipticity was converted
to mean residue ellipticity ([θ]R) using the equation: [θ]R ) θ/(10
× C × Np × l) where θ is the ellipticity in millidegrees, C is the
peptide molar concentration (M), l is the cell path length (cm), and
Np is the number of peptide residues.
of activity (10-fold) and hydrophobicity (see Table 1 and Figure
5 c and d). Such a change in hydrophobicity in combination
with small changes in bond length and torsion angle are likely
to strengthen hydrophobic interactions between the diselenide
bond and the two tyrosine residues or in changing in the binding
orientation, which subsequently increases the hydrophobic
contacts with conserved aromatic residues in the binding site.
Another anticipated advantage of selenoconotoxins is their
higher stability in reducing environments due to the lower redox
potential of the diselenide.18,19,21 RP-HPLC and LC-MS
studies on AuIB and the R-selenoconotoxin Sec[1,3]-AuIB and
Sec[2,4]-AuIB performed in solutions containing equimolar
glutathione or rat plasma at physiological pH demonstrated that
a single diselenide bond was able to largely suppress scrambling
(see Figure 4). However, the selenium analogs showed a similar
half-life to the native peptides in plasma, confirming that the
main degradation occurs mostly via enzymatic digestion. In
order to improve the plasma stability of this class of peptides
even more, N- to C-terminal cyclization is currently the method
of choice and significant progress has been achieved in this field
recently.38,39
In summary, we have developed a novel, highly scalable
method amenable to high throughput R-conotoxin synthesis that
employs resin-supported selenium-chemistry to solve a long-
standing folding problem of an important class of subtype-
selective nAChR antagonists. Stability studies and electrophys-
iological analysis revealed that the selenium analogs of
R-conotoxins are more potent and stable than their native
counterparts. Comprehensive structural analysis showed that the
slightly larger atomic radius of selenium has no significant
impact on the overall structure. Surface analysis and investiga-
tion of the binding pocket of AChBP revealed that the increase
in hydrophobicity of the diselenide bond is likely to be the
reason for observed increase in potency of this toxin class. This
methodology is highly complementary to native chemical
ligation and orthogonal thiol-protecting group strategies and is
anticipated to provide access to more complex disulfide rich
peptides or proteins with similar folding challenges.19,35
4.5. NMR Spectroscopy. NMR spectra were recorded at 290
K on a Bruker Avance 600 MHz spectrometer and processed using
Topspin (Bruker Corp. Billerica, MA, USA) software. The con-
1
centration for the H NMR measurements was ∼1 mM peptide in
90% H2O/ 10% D2O (v/v) at pH 3. 2D NMR spectra were recorded
in phase-sensitive mode using time-proportional phase incremen-
tation for quadrature detection in the t1 dimension.41 The 2D
experiments consisted of a TOCSY using a MLEV-17 spin lock
sequence42 with a mixing time of 80 ms, and NOESY with a mixing
time of 250 ms. Solvent suppression was achieved using a modified
WATERGATE sequence. Spectra were acquired over 6024 Hz with
4096 complex data points in F2 and 512 increments in the F1
dimension. The t1 dimension was zero-filled to 1024 real data points,
and 90° phase-shifted sine bell window functions were applied prior
to Fourier transformation. Chemical shifts were referenced to
internal 2, 2-dimethyl-2-silapentane- 5-sulfonate.
4. Methods
4.1. Peptide Synthesis. All peptides were assembled by manual
Boc-SPPS using HBTU-mediated in situ neutralization protocol
with DMF as solvent.23 Deprotection of the 2,4-dinitrophenyl (Dnp)
group of histidine was carried out prior to HF treatment with 20%
2-mercaptoethanol/ 10% DIEA/ DMF. HF deprotection or cleavage
was performed by treatment of the dried peptide resin (300 mg)
with 10 mL HF/p-cresol/p-thio-cresol (18:1:1, v/v/v) for 2 h at 0
°C. Following evaporation of the HF, the peptides were precipitated
and washed with cold ether, filtered, and either redissolved in 30
mL of 50% ACN/1% TFA and lyophilized, or redissolved directly
in 0.1 M NH4HCO3 (pH 8.4, c ) 0.1 µM) for direct oxidation.
Oxidation was monitored by RP-HPLC, LC-MS and MS, and
the peptides were isolated using preparative C18 RP-HPLC.
4.2. On-Resin Folding using a Safety-Catch Acid Labile
(SCAL) Linker. Peptide assembly was achieved by manual Boc-
SPPS23 using the Fmoc-SCAL linker and a three glycine spacer
between the linker and the aminomethyl ChemMatrix resin.
Deprotection of the Fmoc group of the SCAL linker was performed
with 2× 1 min treatment of 50% piperidine/DMF. 100-500 mg
of the individual peptides on-resin were transferred into labeled
4.6. Glutathione Stability Assay. Peptide samples (0.3 mM)
were dissolved in a solution containing 0.3 mM reduced glutathione
(Sigma Aldrich) in 100 mM phosphate buffer, pH 7.2 and incubated
at 37 °C. Aliquots (30 mL) were taken at different time points,
quenched with extraction buffer (70 mL) consisting of 50% aqueous
acetonitrile, 100 mM NaCl and 1% TFA, and analyzed by
RP-HPLC and LC-MS.21
4.7. Rat Plasma Stability Assay. Rat plasma (Sigma Aldrich)
was incubated at 37 °C for 30 min and 300 mL of plasma was added
to 50 mL of 0.3 mM peptide sample in 100 mM phosphate buffer,
pH 7.2. The samples were incubated at 37 °C and aliquots (30 mL)
were taken at different time points, quenched with extraction buffer
(40) Clark, R. J.; Fischer, H.; Nevin, S. T.; Adams, D. J.; Craik, D. J.
J. Biol. Chem. 2006, 281, 23254–23263.
(41) Marion, D.; Wu¨thrich, K. Biochem. Biophys. Res. Commun. 1983, 113,
967–974.
(38) Clark, R. J.; Fischer, H.; Dempster, L.; Daly, N. L.; Rosengren, K. J.;
Nevin, S. T.; Meunier, F. A.; Adams, D. J.; Craik, D. J. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 13767–13772.
(42) Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 65, 355–360.
(43) Azam, L.; Dowell, C.; Watkins, M.; Stitzel, J. A.; Olivera, B. M.;
McIntosh, J. M. J. Biol. Chem. 2005, 280, 80–87.
(39) Craik, D. J.; Adams, D. J. ACS Chem. Biol. 2007, 2, 457–468.
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Muttenthaler et al.
(70 mL) consisting of 50% aqueous acetonitrile, 100 mM NaCl, and
1% TFA, chilled on ice for 5 min prior to centrifugation at 14 000
rpm for 10 min and analyzed by RP-HPLC and LC-MS.21
Supporting Information Available: Synthetic procedures and
analytical data; general materials and methods; details on the
safety-catch acid labile (SCAL) linker; additional experiments
and conditions on selenium-directed folding; X-ray analysis;
details on biological analysis. This material is available free of
charge via the Internet at http://pubs.acs.org.
Acknowledgment. We acknowledge Dr. Aline Dantas de
Araujo for her contribution to the synthetic work and Alun Jones
for the help with MS. This research was financially supported by
an Australian Research Discovery Project Grant and a National
Health and Medical Research Council Program Grant. D.J.C. is an
NHMRC Professorial Fellow.
JA910602H
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