Synthesis of K48-linked diubiquitin using dual native chemical ligation at lysine

Article abstract of DOI:10.1039/c0cc01382j

The dual native chemical ligation at lysine strategy was revised by replacing the acid-labile Cbz protecting group with photolabile NVOC at the 4-mercaptolysine side chain. The optimized strategy was subsequently applied to the synthesis of K48-linked diu

Full text of DOI:10.1039/c0cc01382j

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Synthesis of K48-linked diubiquitin using dual native chemical ligation at
lysinew
a
b
Renliang Yang,z Kalyan Kumar Pasunooti,z Fupeng Li,a Xue-Wei Liu*b and Chuan-Fa Liu*a
Received 13th May 2010, Accepted 5th August 2010
DOI: 10.1039/c0cc01382j
The dual native chemical ligation at lysine strategy was revised
by replacing the acid-labile Cbz protecting group with photo-
labile NVOC at the 4-mercaptolysine side chain. The optimized
strategy was subsequently applied to the synthesis of K48-linked
diubiquitin.
employing a disulfide linkage was also reported.11 These two
forms of non-native monoubiquitination were shown to exert
a similar effect to the native form.
Previously, we reported the synthesis of a monoubiquitinated
peptide through dual native chemical ligation at lysine.8a,12
A
lysine derivative with a thiol group at C-4 of the lysine side
chain was used to mediate the chemical ligation at both the
a- and e-amino group of lysine. While the ligation at the
a-amino group enables the chemical synthesis of the target
protein itself, the side chain ligation can be used for the
site-specific installation of the ubiquitin tag. In the previous
study, we employed benzyloxycarbonyl (Cbz) as the orthogonal
protecting group for the e-amino group of 4-mercaptolysine to
make sure that the ligation first selectively occurred at the
a-amine. Then the Cbz group was removed with a strong acid,
such as trifluoromethanesulfonic acid (TFMSA), to free the
e-amine for the side chain ligation. We realize that the harsh
conditions for Cbz removal may limit the application scope of
our methodology for large protein ubiquitination. In this
communication, we revise our orthogonal protection strategy
by replacing Cbz with a photolabile protection group,
o-nitroveratryloxycarbonyl (NVOC) (Fig. 1). We then
apply our revised approach for the synthesis of K48-linked
diubiquitin.
Ubiquitination is one of the most important protein post-
translational modifications.1 It refers to the linking of the
C-terminal glycine of the ubiquitin protein (76 amino acids) to
the lysine side chain of the modified proteins. Proteins can
undergo monoubiquitination or polyubiquitination.1 As a
well-known example of monoubiquitination, the modification
at the C-terminal region of histone H2A is associated with
gene silencing.2 The monoubiquitination at the C-terminus
of H2B was shown to be associated with transcription
elongation.3 In the case of polyubiquitination, a chain consisting
of multiple ubiquitins with defined linkage is attached to the
lysine side chain of a modified protein target.4 Ubiquitin
contains seven lysine residues (K6, 11, 27, 29, 33, 48, 63)
and each of these lysine residues can be employed for ubiquitin
chain formation. The biological roles of polyubiquitin may be
related to its linkage specificity.4 K48-linked polyubiquitin is
the best understood polyubiquitination and is shown to serve
as a signal for targeting the protein to proteasomal degradation.5
In contrast, K63-linked polyubiquitination is involved in a
signaling pathway and associated with kinase activation.6 To
elucidate the biological role of ubiquitination, it is important
to isolate a sufficient amount of ubiquitinated proteins for
in vitro study. In eukaryotes, ubiquitination is catalyzed by
three classes of enzymes, ubiquitin-activating enzymes E1,
ubiquitin-conjugating enzymes E2 and ubiquitin ligase E3.1
In vitro enzymatic synthesis of ubiquitinated proteins is limited
by the requirement for identifying the ligases and the availability
of the ligase. In recent years, chemical ubiquitination was
succeeded by either Na-auxiliary-mediated7 or mercaptolysine-
mediated8 peptide ubiquitination. These methods generate the
native Gly76–Lys(e) isopeptide bond which is typical for
ubiquitination. Cysteine mediated ligation/desulfurization
enables the synthesis of Gly76Ala monoubiquitinated proteins.9
Essentially, all these methods are based on the native chemical
ligation (NCL) approach.10 Recently, chemical ubiquitination
The Ne-NVOC protected 4-mercaptolysine derivative 1 was
synthesized in a similar way to that previously reported8a by
using NVOC-Cl instead of Cbz-Cl as the side chain protecting
reagent (For the details, see Electronic Supplementary
Information).
The overall strategy of synthesizing the diubiquitin is
shown in Scheme 1. To synthesize K48-linked diubiquitin (8)
using the dual NCL at lysine method, the crucial step is
the installation of 4-mercaptolysine at position 48 of the
monoubiquitin. The synthesis of ubiquitin and its mutants
through stepwise synthesis or sequential chemical ligation/
desulfurization has been reported.13 Here, we synthesized the
K48(4-SH)-containing ubiquitin (6) using C-to-N sequential
ligation with Ala28Cys and K48(4-SH) as the ligation
junctions. After we got 6, the NVOC group was removed by
365 nm UV irradiation. Monoubiquitin 7 with free e-NH2 on
K48 was then reacted with ubiquitin thioester Ub(1–76)-MES
a Division of Chemical Biology and Biotechnology, School of
Biological Sciences, Nanyang Technological University,
60 Nanyang Drive Singapore 637551. E-mail: cfliu@ntu.edu.sg;
Fax: (+65) 6791-3856
b Division of Chemistry and Biological Chemistry, School of Physical
and Mathematical Sciences, Nanyang Technological University,
Singapore. E-mail: xuewei@ntu.edu.sg; Fax: (+65) 6791-3856
w Electronic supplementary information (ESI) available: Experimental
procedure, MS data and NMR data. See DOI: 10.1039/c0cc01382j
z These authors contributed equally to this work.
Fig. 1 Ne-NVOC protected 4-mercaptolysine derivative.
Chem. Commun., 2010, 46, 7199–7201 7199
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Fig. 2 C8 analytic HPLC data for the synthesis of 6. (A) and (B):
Ligation between 3 and 4 at 10 min and 12 h, respectively. Peak a1, the
1 : 10 mixture of 3 and its MES thioester; peak b, reduced 4; peak c,
ligation product Thz28-K48(4-SH, Ne-NVOC)-G76-OH; peak a2,
about 1 : 1 mixture of 3 and its MES thioester. (C): The in situ
conversion of Thz to Cys for 4.5 h. peak a3, peptide 3; peak a4,
Cys28-G47-NHOCH3; peak d, product 5. (D): Purified 5. (E): Ligation
between 2 and 5 overnight. Peak e, the MES thioester of 2; peak f,
cyclization product of 2; peak g, ligation product 6. (F): Purified 6.
Scheme
1
The strategy used for the synthesis of K48-linked
diubiquitin. Note: the Met1 of the synthesized ubiquitin was changed
to Leu to avoid the oxidation.
which was generated by thiolysis of ubiquitin–intein fusion
protein with sodium mercaptoethanesulfonate (MESNa). To
generate the native K48-linked diubiquitin, free radical
mediated desulfurization14 was performed for the ligation
product to convert Cys28 to Ala and K48(4-SH) to Lys.
HPLC data for the synthesis of 6 are shown in Fig. 2. The
middle segment of ubiquitin, peptide athioester 3, was reacted
with C-terminal segment 4 through 4-mercaptolysine mediated
ligation. 8 mg of 3 and 10 mg of 4 were dissolved in ligation
buffer (6 M GdnꢀHCl, 0.2 M phosphate, 20 mM TCEP, pH 7.5).
In the presence of TCEP, the disulfide in 4 was immediately
reduced and gave the free thiol. Upon addition of the thiol
additive MESNa (0.2 M), 3 was converted to its MES thioester.
The ligation proceeded efficiently. After 12 h, the reaction was
completed. The conversion of Thz (1,3-thiazolidine-4-carboxo
group) to Cys was directly performed in the ligation mixture.
0.4 M MeONH2ꢀHCl (final concentration) was added into the
mixture and the pH was adjusted to 4.0. After 4.5 h at room
temperature, the deprotection was completed (Fig. 2C).
Product 5 was purified by C18 semi-preparative HPLC. About
10 mg of ligation product was obtained after lyophilization.
For the next ligation step, 7.3 mg of peptide thioester 2 and
10 mg of 5 were dissolved in the ligation buffer (6 M GdnꢀHCl,
0.2 M phosphate, 0.2 M MESNa, 20 mM TCEP, pH 7.5). The
reaction was completed overnight. The major side reaction
was the self-cyclization of 2 involving possibly the C-terminal
lysine residue. The full-length ubiquitin 6 was purified by C18
semi-preparative HPLC. About 5.1 mg of 6 was obtained after
lyophilization.
ionization mass spectrometry (ESI-MS). After 2 h, ESI-MS
confirmed that the NVOC group was completely removed
to afford product 7 (Fig. 3). The solution was diluted
and subjected to C18 semi-preparative HPLC purification.
1.3 mg of 7 was obtained after lyophilization.
To synthesize K48-linked diubiquitin, ubiquitin 7 was
reacted with Ub(1–76)-MES (Fig. 4). About 1.3 mg of 7 and
1.5 mg of Ub(1–76)-MES were dissolved in 150 mL ligation
buffer (6 M GdnꢀHCl, 0.1 M phosphate, 40 mM TCEP, 1%
v/v benzyl mercaptan, pH 8.0). After 6 h, the ligation product
was formed with a yield of about 65% based on analytic
HPLC. After another 4 h, the yield of ligation product
increased slightly. 0.8 mg of ligation product was isolated
after purification by HPLC.
To get the native K48-linked diubiquitin, free radical
mediated desulfurization was performed to convert cysteine
28 and 4-mercaptolysine 48 at the ligation junctions to alanine
and lysine, respectively. After 9 h of treatment with the free
radical initiator VA-044, both sulfur atoms on the two residues
were removed, as confirmed by ESI-MS (Fig. 5C). Deconvoluted
ESI-MS showed the correct molecular weight of the final
To remove the photolabile protection group, 1.7 mg
of 6 was dissolved in 60% acetonitrile aqueous solution
(containing 0.045% trifluoroacetic acid) at a concentration of
1 mg mLꢁ1. The solution was irradiated with 365 nm UV light.
The deprotection process was monitored with electrospray
Fig.
3
ESI-MS monitored the removal of the NVOC group.
Left panel: before UV irradiation, calculated M = 8850.0 Da,
deconvoluted M = 8850.8 Da; right panel: UV irradiation for 2 h,
calculated M = 8610.8 Da, deconvoluted M = 8612.6 Da.
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the one in Coomassie blue staining (Fig. 5B). To test whether
the synthesized diubiquitin can be folded to its native
form, circular dichroism (CD) was measured with dialyzed
diubiquitin 8. The CD spectrum indicated that the diubiquitin
was well folded after dialysis (Fig. 5D). Next we performed the
cleavage assay using ubiquitin C-terminal hydrolase. It was
found that the diubiquitin can be hydrolyzed by the hydrolase,
UCH-L3 (Fig. S11, Electronic Supplementary Information).
In summary, herein we have optimized the dual native
chemical ligation approach by replacing the strong acid-labile
Cbz with the photolabile NVOC protecting group for the
side-chain amine of 4-mercaptolysine. The synthesis of
K48-linked diubiquitin using the improved protocol demon-
strates the practical utility of this ligation strategy in synthetic
protein chemistry.
Fig.
4 C4 analytic HPLC monitored ligation between 7 and
Ub(1–76)-MES at 0 h, 6 h and 10 h, respectively. Peak a: mixture of
7 and ubi(1–76)-MES. Peak b: ligation product. Peak c: mixture of
ubi(1–76)-OH and small amount of remaining 7 and ubi(1–76)-SBn.
Peak *: nonproteinous product. Note: 0 h HPLC was run before
adding benzyl mercaptan. Bn = benzyl.
This work was supported by A*Star of Singapore (BMRC
08/1/22/19/588 to C.-F.L.) and Nanyang Technological
University.
Notes and references
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Fig. 5 Characterization of the synthesized K48-linked diubiquitin.
(A) C4 analytic HPLC. (B) 18% SDS-PAGE analysis stained with
Coomassie blue (left) and Western blot (right). M, protein marker;
Lane 1, monoubiquitin; Lane 2, K48-linked diubiquitin 8. (C) ESI-MS
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native K48-linked diubiquitin. For the characterization
of the final product, the diubiquitin was checked with 18%
SDS-PAGE. Coomassie blue staining showed a single band.
Western blot with antibody FK2H, which is an HRP-
conjugated antibody against mono- and polyubiquitinated
conjugates but not free ubiquitin, detected the same band as
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 7199–7201 7201