Chemical Synthesis of Native S-Palmitoylated Membrane Proteins through Removable-Backbone-Modification-Assisted Ser/Thr Ligation

Article abstract of DOI:10.1002/anie.201914836

The preparation of native S-palmitoylated (S-palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S-palm membrane proteins by removable-backbone-modification-assisted Ser/Thr ligation (RBM^GABA-assisted STL). This method involves two critical steps: 1) synthesis of S-palm peptides by a new γ-aminobutyric acid based RBM (RBM^GABA) strategy, and 2) ligation of the S-palm RBM-modified peptides to give the desired S-palm product by the STL method. The utility of the RBM^GABA-assisted STL method was demonstrated by the synthesis of rabbit S-palm sarcolipin (SLN) and S-palm matrix-2 (M2) ion channel. The synthesis of S-palm membrane proteins highlights the importance of developing non-NCL methods for chemical protein synthesis.

Full text of DOI:10.1002/anie.201914836

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Chemical Synthesis of Native S-palmitoylated Membrane
Proteins through Reversible Backbone Modification Assisted Ser/
Thr Ligation
Authors: Dong-Liang Huang, Cédric Montigny, Yong Zheng, Veronica
Beswick, Ying Li, Xiu-Xiu Cao, Thomas Barbot, Christine
Jaxel, Jun Liang, Min Xue, Chang-Lin Tian, Nadège Jamin,
and Ji-Shen Zheng
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201914836
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10.1002/anie.201914836
Angewandte Chemie International Edition
DOI: 10.1002/anie.2019 ((will be filled in by the editorial staff))
Chemical Protein Synthesis
Chemical Synthesis of Native S-palmitoylated Membrane Proteins
through Reversible Backbone Modification Assisted Ser/Thr
Ligation
Dong-Liang Huang,[a,+] Cédric Montigny,[b,+] Yong Zheng,[a,+] Veronica Beswick,[b,c] Ying Li,[a] Xiu-Xiu
Cao,[a] Thomas Barbot,[b] Christine Jaxel,[b] Jun Liang, [a] Min Xue, [a] Chang-Lin Tian,[a] Nadège Jamin,[b]
Ji-Shen Zheng[a*]
Abstract: Preparation of native S-palmitoylated (S-palm) membrane
proteins represents one of the unsolved challenges in chemical protein
synthesis. Herein, we reported the first chemical synthesis of S-palm
membrane proteins by reversible backbone modification assisted
Ser/Thr Ligation (RBMGABA-assisted STL). This method involves two
critical steps: (1) Synthesis of S-palm peptides by a new γ-aminobutyric
acid group-based RBM (RBMGABA) strategy, (2) Ligation of the S-palm
RBM-modified peptides to give the desired S-palm product by STL. The
utility of RBMGABA-assisted STL method was demonstrated by the
synthesis of rabbit S-palm sarcolipin (SLN) and the S-palm matrix-2
(M2) ion channel. The synthesis of S-palm membrane proteins
highlights the importance of developing non-NCL methods for chemical
protein synthesis.
synthesize a number of proteins5 (e.g. betatrophin, interleukin 25,
and HMGA1a) which, however, can in principle also be obtained
by NCL-based methods. Examples remain to be demonstrated
where the non-NCL methods must be used to accomplish the
chemical synthesis of certain practically important proteins, and
one such case would be S-palmitoylated proteins.
S-palmitoylation refers to the covalent modification of palmitic
acyl onto a Cys residue of protein substrates via a thioester bond.
This modification is abundant in hydrophobic membrane proteins,
and plays key roles in many biological processes such as, protein
localization, signal transduction, immune response, and cell
apoptosis.6 Dysfunction of S-palmitoylation can cause neurological
diseases, immune diseases or cancers. For example,
depalmitoylation of melanocortin-1 receptor (MC1R) stimulates
melanogenesis.7 S-palmitoylation on programmed death ligand-1
(PD-L1) promotes tumor growth.8 Structure-function relationships
of S-palmitoylated membrane proteins need to be studied to
decipher the role of S-palmitoylation and facilitate drug discovery.9
Additionally, S-palmitoylated membrane protein probes are
requisite tools for screening of substrate-specific depalmitoylase
inhibitors.6a,10 In this context, it is important to produce
homogeneous samples of S-palmitoylated membrane proteins in
workable quantities. Recent studies showed that the commonly
used solid-phase peptide synthesis (SPPS) can only afford short
S-palmitoylated peptides.11 On the other hand, although
conjugation methods (e.g. maleimidocaproyl conjugation, Diels-
Alder ligation) were used to generate some S-palmitoylated protein
analogues, the products contained non-native structural motifs.12
To date, there has been no study on the preparation of native S-
palmitoylated membrane proteins by chemical ligation methods.13
Herein, we reported the first chemical synthesis of native S-
palmitoylated membrane proteins using a newly developed
removable backbone modification assisted serine/threonine
ligation (RBMGABA-assisted STL) strategy. This work demonstrates
the importance of integrating different modern techniques to
overcome the increasingly more difficult challenges in chemical
protein synthesis. A critical problem encountered in our study is the
incompatibility between the previous RBM method and S-
palmitoylation as well as STL ligation. To meet this challenge, a
new auto-cyclization γ-aminobutyric acid (GABA)-based RBM
(RBMGABA) strategy was developed and found to be critical to the
success of RBMGABA-assisted STL ligation (Scheme 1). The
effectiveness of RBMGABA-assisted STL was examined through the
synthesis of two practically important native S-palmitoylated
membrane proteins, namely, rabbit S-palmitoylated sarcolipin (S-
palm SLN) and S-palmitoylated matrix-2 (S-palm M2) ion channel
from Influenza A virus. Thus, our work provides examples where
non-NCL methods play an irreplaceable role in the chemical
synthesis of some important protein targets.
Introduction
Chemical protein synthesis provides access to preparation of
custom-made proteins, such as site-specifically post-translationally
modified proteins and mirror-image proteins for their biochemical,
biophysical and pharmaceutical studies.1 Native chemical ligation
(NCL), in which a C-terminal peptide thioester reacts with an N-
terminal Cys peptide to form a native amide linkage, is the most
popular method for chemical protein synthesis.2 NCL requires an
N-terminal Cys to mediate ligation but the natural abundance of
Cys is low (~1.8%). To expand the scope of NCL, a series of
advanced versions of NCL have been developed for the ligation at
non-Cys residues, such as Ala, Lys, Val, Ser and Thr.3 On the other
hand, some non-NCL methods4 have also been developed, such
as serine/threonine ligation (STL)4b,4c and α-keto acid-
hydroxylamine (KAHA) ligation4d,4e, to enable peptide ligation at
non-Cys sites. These non-NCL methods have been used to
[a]
[b]
High Magnetic Field Laboratory, Chinese Academy of Sciences,
and Hefei National Laboratory for Physics Sciences at Microscale,
School of Life Sciences, University of Science and Technology of
China, Hefei 230027, China
E-mail: jszheng@ustc.edu.cn
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS,
Univ. Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette
cedex, France
[c]
[+]
Department of Physics, Evry-Val-d’Essonne University, F-91025
Evry, France
These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
1
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10.1002/anie.201914836
Angewandte Chemie International Edition
Scheme 1. The RBMGABA-assisted STL strategy for chemical synthesis of native
S-palmitoylated membrane proteins. Parts of the membrane protein are
represented by blue frames. An is any primary amino acid of the membrane
protein sequence. R represents H or CH3 group.
Figure 1. The synthesis of the full-length S-palm SLN by Fmoc SPPS. (a) Amino
acid sequence of S-palm SLN. The hydrophobic residues are colored in green.
(b) Solubility and HPLC profile of the crude native S-palm SLN 1 in 50 %
CH3CN/H2O . (c) HPLC traces of crude RBM-modified S-palm SLN samples
synthesized by different methods. The non-peptide peak was highlighted with *.
Results and Discussion
with a RBM tag at Leu25 using the reported protocol.22 The crude
RBM-modified peptide was found to be well soluble, but its crude
RP-HPLC profile was too complex to identify any target peptide
(Fig. 1c). To improve the purity of crude S-palm SLN, we tried
many synthetic protocols (for example, NMP/DMF co-solvents,
increasing the temperature, microwave, pseudoproline, changing
the RBM’s installing site and installing two RBM tags) during the
peptide assembly, but there was no obvious improvement (Fig. 1c
and Supplementary Fig. S3). These results indicated that the
desired full-length RBM-modified S-palm SLN cannot be directly
obtained by Fmoc SPPS. This may be attributed to the fact that
SLN contains lots of sterically hindered and hydrophobic amino
acids, which makes coupling/deprotection steps incomplete during
peptide assembly. In addition, S-palmitoylation further increases
the synthetic complexity.11
Chemical synthesis of S-palm SLN by Fmoc SPPS
Our work started with the synthesis of S-palm SLN, a 31-
residue single transmembrane protein, for its biochemical and
biophysical studies (Fig. 1a).14 SLN regulates Ca2+ transport
activity of SERCA1a to control the intracellular calcium
homeostasis and muscle contraction/relaxation. Dysfunction of
SLN results in non-shivering thermogenesis and heart failure and
therefore, SLN is considered as a potential drug target.15 N- and C-
terminus of SLN are located within the cytosol and the lumen
respectively.16 Recently, native rabbit SLN was found to be S-
palmitoylated on Cys9 and it was suggested that S-
palmitoylation/depalmitoylation of SLN might regulate SERCA1a
activity.17 To understand the detailed regulatory mechanism of S-
palm SLN on SERCA1a activity at a molecular level, the S-palm
SLN sample is required to have available isoforms from the same
species and control of the stoichiometry during catalysis. 18
Overall, our attempts to synthesize S-Palm SLN showed that
this family of proteins is extraordinarily difficult to produce and
therefore, posing an interesting and practically important challenge
for modern chemical protein synthesis.
To prepare the full-length S-palm SLN 1, we initially employed
direct Fmoc solid-phase peptide synthesis (SPPS). In the SPPS,
the 4-monomethoxytrityl (Mmt) protected Cys9 was installed into
the peptide. After peptide assembly, the Mmt group in the resin-
bound peptide precursor was selectively deprotected by treating
with 2% trifluoroacetic acid (TFA) cocktails to give a free thiol.19
The released thiol then reacted with palmitic anhydride to generate
S-palm SLN 1. Unfortunately, the crude peptide 1 was found to be
insoluble and no target product was detected on the reverse-phase
high-performance liquid chromatography (RP-HPLC) profile (Fig.
1b). We also examined other previously developed strategies20
(such as NMP/DMF co-solvents, microwave, pseudoproline, O-
acyl isopeptide and attachment of solubilizing tags into peptide side
chains) to improve the synthetic efficiency. However, no significant
improvement was observed on RP-HPLC (Supplementary Fig.
S1), presumably due to the strong hydrophobicity of 1.
One effective solution to improve the solubility of very poorly-
soluble proteins is our removable backbone modification (RBM)
strategy which enables installation of RBM groups into backbone
amides and increases the solubility by both addition of solubilizing
tag and disruption of aggregation to effectively facilitate the
chemical synthesis of a number of membrane proteins.21 Using the
RBM strategy, we synthesized the S-palm SLN(L25,RBM,C9,palm) 2
Development of the RBMGABA-assisted STL method for the
chemical synthesis of S-palm SLN
After failure of direct synthesis of S-palm SLN by Fmoc SPPS,
we turned to peptide ligation. However, the previous work23 and
our model NCL ligation of S-palmitoylated peptides
(Supplementary Fig. S11-S13) showed that the S-palmitoylated
peptide contains a reactive S-palmitoyl thioester group which is
labile towards the N-terminal Cys of peptides in NCL, making the
most popular NCL incompatible with S-palmitoylation (Fig. 2a).
Therefore, the non-NCL ligation methods must be used for the
synthesis of S-palm SLN, among which we chose STL ligation. In
STL, an N-terminal Ser/Thr peptide and
a
C-terminal
salicylaldehyde (SAL) ester peptide can undergo a chemoselective
capture and a subsequent O-to-N acyl transfer to afford an N,O-
benzylidene acetal-linked intermediate, which can be quantitatively
converted to
a
native amide bond upon acidolysis.4c We
hypothesized that the SAL ester and Ser/Thr groups would be
compatible with S-palmitoylation. To implement the idea, S-palm
SLN was divided into two peptides, a S-palmitoylated SAL ester
peptide SLN(1-12,C9,Palm)-SAL 3 and an N-terminal Thr peptide
2
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10.1002/anie.201914836
Angewandte Chemie International Edition
Figure 2. Chemical synthesis of S-palm SLN by RBMGABA-based STL method. (a) The incompatibility of S-palmitoylation with NCL. (b) The RBMGABA-based STL
method for the synthesis of 1. (c) The incompatibility of S-palmitoylation with the previous RBM strategy. (d) The synthesis of RBM-modified S-palmitoylated 5 by the
new RBMGABA strategy. (e) HPLC traces of the crude S-palmitoylated peptide 5’ and the removal of GABA group of 5’ to obtain 5. (f) ESI-MS data of S-palmitoylated
peptides 5’, 5 and the ligation product 7. (g) HPLC traces of STL between 5 and 6 to afford 7. (h) MALDI-TOF MS traces of the cleavage of RBM tags of 7 to give 1.
(i) Tricine-SDS-PAGE of the cleavage of RBM tags of 7 to give 1.
SLN(13-31) 4. We tried to prepare peptides 3 and 4 through Fmoc
SPPS. However, even though peptide 3 could be detected by
analytical RP-HPLC, its isolated yield (<1%) was very low. Even
worse, the other peptide 4 could not be detected in the crude
peptide that was a complex mixture of insoluble materials.
compatibility problem between the RBM method and S-
palmitoylation as well as STL.
To develop an effective method of RBM-assisted STL, we
screened different designs by using intramolecular cyclization for
deprotection. For example, we tested our previously used N-
methyl-N-[2-(methylamino)ethyl]carbamoyl group, which was
reported to undergo auto-cyclization at weakly alkaline pH.21a
However, the test results showed that the deprotection conditions
of this group caused serious hydrolysis of S-palmitoylation
(Supplementary Fig. S14). After several trials, we turned to a
previous finding of the remarkable self-cyclization property of the
amino carboxyl ester structures under weakly acidic reaction
conditions24. Thus the γ-aminobutyric acid (GABA) group was
developed to mask the 2-OH group in RBM to ensure its stability
towards TFA used in SPPS (Supplementary Fig. S16-S19). As
shown in Fig. 2d, GABA was coupled with the phenolic hydroxyl
group to give the phenolic ester on RBM. Subsequent S-
palmitoylation and coupling of SAL ester were successful. More
importantly, after TFA cleavage, the target peptide SLN(1-
12,E2,RBM-OGABA,C9,Palm)-SAL 5’ was obtained as a main peak from
the RP-HPLC (Fig. 2e). 5’ was characterized by ESI-MS and
purified by semi-preparative RP-HPLC using the water-acetonitrile
mobile phase with an isolated yield of 11 %.
To solve the problem, we proposed to use the strategies of
incorporation of a solubilizing tag into peptide side chains or C-/N-
terminus20, such as Lys-based Ddae-type tag20b and Cys-based
Trt-/Phacm-type tag20c,20d. However, most of these strategies
cannot be employed for the synthesis of S-palm SLN due to the
lack of amino acid sites to install tags (no additional Cys and Lys)
or the incompatibility of S-palmitoylation due to the use of
nucleophilic conditions (e.g. high pH, heavy metals and thiol
reagents) in the final tag’s removal step. Therefore, we decided to
use the RBM strategy and install RBM tags into the S-palmitoylated
SAL ester peptide SLN(1-12,E2,RBM,C9,Palm)-SAL 5 and Thr peptide
SLN(13-31,L16,RBM) 6 at Glu2 and Leu16, respectively (Fig. 2b).
The acetylated peptide precursor SLN(1-12,E2,RBM-OAc,C9,Palm)-SAL
5’’ was obtained smoothly, but we soon realized that the activation
of RBM through our previous de-acylation strategy could not be
accomplished due to the presence of both S-palmitoyl thioester
and SAL ester groups (Fig. 2c). Thus, a critical challenge for the
development of RBM-assisted STL is how to overcome the
3
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10.1002/anie.201914836
Angewandte Chemie International Edition
Next, we tested the critical step of RBM activation by the
removal of the acyl group at its 2-OH site. Thus, purified 5’ was
dissolved into pyridine/AcOH (7/6, mol/mol) conditions that was
needed for carrying out STL. The RP-HPLC traces showed that the
auto-cyclization was smooth under the weakly acidic conditions of
STL and could be completed within 5 min to give the target peptide
5 in a 77% isolated yield (Fig. 2e). The results showed that the
GABA-based RBM strategy was fully compatible with S-palmitoyl
and SAL ester groups and could be activated in the special reaction
conditions of STL (namely pyridine/AcOH) (Supplementary Fig.
S20-23). Furthermore, using the same RBMGABA strategy, the other
RBM-modified peptide 6 was also easily obtained with a 21%
isolated yield (Supplementary Fig. S27).
The STL ligation of 5 (11.2 mg) and 6 (13.0 mg) was then
carried out in 0.2 mL of pyridine/acetic acid buffer solution (1/6,
mol/mol) at 30 oC. Analytical HPLC traces showed that the reaction
took place smoothly within 4 h to give the N,O-benzylidene acetal
intermediate 7 (Fig. 2g). The product 7 was purified by RP-HPLC
(8.5 mg, 35% isolated yield) and characterized by ESI-MS (Fig. 2f).
assisted laser desorption/ionization time of flight mass
spectrometry (MALDI-TOF-MS) traces showed that the N,O-
benzylidene acetal group of 7 was removed to afford 7’ within 5
min and the two RBM tags were quantitatively cleaved within 3 h
(Fig. 2h). The removal of the backbone modification groups was
also characterized through the tricine-sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) analysis,
which gave an expected single band shift before and after
acidolysis treatment (Fig. 2i). After that, the diluted HCl solution
was concentrated by N2 blowing, and then precipitated by cold
diethyl ether to afford the final S-palm SLN 1 with a 55% yield. The
synthetic S-palm SLN was refolded in 10 mM Tris-HCl, pH 7.5, 4%
(w/v) sodium dodecyl sulfate (SDS) at 25 °C and the circular
dichroism (CD) spectrum showed two minima at 208 and 222 nm,
indicating the α-helical structuration (Supplementary Fig. S30).
Chemical synthesis of S-palm M2 by RBMGABA-assisted STL
To further demonstrate the utility of the RBMGABA-based STL
strategy, we synthesized S-palmitoylated M2 ion channel from
influenza A virus. The 97-residue M2 can form homotetramers to
transport protons across the viral envelope. It plays critical roles
during the virus entry, replication and assembly of infectious virus
particles. M2’s functions are regulated by several posttranslational
Finally, the purified peptide
7 was dissolved in 0.1 M
HCl/hexafluoro-isopropanol (HFIP) containing 1% TIPS at room
temperature, an optimized cleavage condition (Supplementary
Fig. S29), to remove the N,O-benzylidene acetal and two RBM
tags.25 The final product 1 could not be monitored by RP-HPLC
because of its strong hydrophobicity. Nonetheless, the matrix-
Figure 3. Chemical synthesis and characterizations of the Cys50-palmitoylated M2 by the RBMGABA-assisted STL method. (a) The synthesis of S-palmitoylated 10 by
RBMGABA strategy. M indicates a replacement of Met with Nle. (b) HPLC traces of crude and purified peptide 10 (up) and ESI-MS of 10 (down). (c) The synthetic route
by RBMGABA-assisted STL. (d) HPLC traces of ligation of 9 and 10. (e) ESI-MS of 11. (f) ESI-MS of 12. (g) Tricine-SDS-PAGE of 11 and 12. (h) CD analysis of synthetic
S-palm M2 in 50mM n-octyl-β-D-glucopyranoside (OG) vesicles.
4
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10.1002/anie.201914836
Angewandte Chemie International Edition
modifications, such as phosphorylation, ubiquitination and S-
palmitoylation.26 Recently, phosphorylated M2 has been
chemically synthesized to investigate the role of phosphorylation
on M2’s functions.21a The synthesis of S-palmitoylated M2 would
also provide unique research tools for the study of the role of M2’s
S-palmitoylation, whereas the reported NCL-based method for
phosphorylated M2 cannot used to prepared S-palmitoylated M2
owing to the incompatibility of NCL with S-palmitoylation.
Acknowledgements
This study was supported by the National Key R&D Program of
China (No. 2019YFA0706900), the National Natural Science
Foundation of China (No. U1732161 and 91753120), the Science
and Technological Fund of Anhui Province for Outstanding Youth
(No. 1808085J04), and the Innovative Program Development
Foundation of Hefei Center Physical Science and Technology (No.
2017FXCX002). The PHC program Xu Guangqi (No. 43357ZD) is
acknowledged for providing travel funds to J.S.Z., C.M. and V.B.
between Heifei and Saclay. This work was supported by the French
Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-
INSB-05). We thank F. Beau for housing the liquid scintillation
counting (CEA/Joliot Institute, France). We thank the Instrument
Analysis Center of University of Science and Technology of China
for assistance on characterizations.
The RBMGABA-assisted STL method was applied to the
preparation of Cys50-palmitoylated M2 (S-palm M2). As shown in
Fig. 3c, S-palm M2 was divided into two segments, M2(1-30)-SAL
9 and M2(31-97,L36,RBM,C50,palm) 10. According to the previous
work5c, pepide 9 was prepared by using Fmoc SPPS, in which Met
was mutated to Norleucine to avoid the possible oxidization during
the peptide synthesis.5e On the other hand, the segment M2(31-
97,C50,palm) without RBM modification could not be prepared by
Fmoc SPPS owing to poor handling property (Supplementary Fig.
S32). When the previous acetyl-based RBM method was used, the
RBM-modified peptide 10 could not be obtained because of the
decomposition of S-palmitoyl groups during the removal of acetyl
groups. When the new RBMGABA strategy was used, pepide 10 was
obtained in 15% isolated yield (Fig. 3a). The purified peptide 10
was well soluble and characterized by RP-HPLC and ESI-MS (Fig.
3b). The two segments were subjected to STL ligation in
pyridine/AcOH to produce the N,O-benzylidene acetal intermediate
11 in 24% isolated yield (Fig. 3d). The ligation intermediate 11 was
well soluble and could be readily purified by RP-HPLC and verified
by ESI-MS (Fig. 3e). Finally, the purified 11 powder was dissolved
into 0.1 M HCl/TFIP containing 1% TIPS at room temperature to
removal the backbone modifications. The solution was
concentrated by N2 blowing, and then precipitated with chilled
diethyl ether to afford the target S-palm M2 12, which was
successfully characterized by ESI-MS and Tricine-SDS-PAGE
analyses, both indicating that the desired product was obtained
(Fig. 3f, 4g). In addition, the CD spectrum of S-palm M2 in the
presence of 50mM n-octyl-β-D-glucopyranoside (OG) showed
characteristic negative bands at 208 and 222 nm (Fig. 3h),
suggesting the folded S-palm M2 with α-helical structures.
Conflict of interest
The authors declare no conflict of interest.
Keywords: Chemical protein synthesis • Serine/threonine ligation
• Removable backbone modification • S-palmitoylation •
Membrane proteins
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Conclusion
In summary, in the present study we show that preparation of
S-palmitoylated membrane proteins poses an important, but very
challenging problem for modern chemical protein synthesis. To
meet the challenge, we developed a RBMGABA-assisted STL
ligation method, which exploits the solubilizing effect of RBM
groups and the compatibility of STL ligation with S-palmitoylation.
Key to the success of the new method is the development of a new
γ-aminobutyric acid (GABA) group in RBM, whose rapid auto-
cyclization property enables its selective activation and removal in
the presence of S-palmitoylation under the STL ligation conditions
(pyridine/AcOH). The effectiveness of RBMGABA-assisted STL
strategy was demonstrated by the successful synthesis of two S-
palmitoylated membrane proteins, namely, sarcolipin and M2.
Access to native S-palmitoylated proteins paves a way to their
biochemistry, biophysics, and pharmaceutics studies. Finally, the
synthesis of S-palmitoylated membrane proteins provides
examples to show the importance of developing non-NCL methods
for chemical protein synthesis.
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6
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10.1002/anie.201914836
Angewandte Chemie International Edition
Chemical Protein Synthesis
Dong-Liang Huang, Cédric Montigny,
Yong Zheng, Veronica Beswick, Ying Li,
Xiu-Xiu Cao, Thomas Barbot, Christine
Jaxel, Jun Liang, Chang-Lin Tian,
Nadège Jamin, Ji-Shen Zheng
Chemical Synthesis of Native S-
palmitoylated Membrane Proteins
through Reversible Backbone
Modification Assisted Ser/Thr
Ligation
RBMGABA-assisted STL strategy (removable backbone modification-assisted
serine/threonine ligation) was developed for the chemical synthesis of native S-
palmitoylated membrane proteins. The successful synthesis of S-palmitoylated
membrane proteins (e.g. S-palmitoylated sarcolipin and M2) for the first time
demonstrated the importance of non-NCL methods in chemical protein synthesis.
7
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