Expressed protein ligation at methionine: N-terminal attachment of homocysteine, ligation, and masking

Article abstract of DOI:10.1002/anie.201302065

A useful handle: One major limitation of protein semi-synthesis is the need for Cys at the ligation site in native chemical ligation reactions. It is shown that a transferase enzyme can deliver homocysteine to the N-terminus of an expressed protein (see scheme). Homocysteine can be used in a ligation reaction and then converted to Met. This allows one to use the MetArg or MetLys motif as a point of disconnection in semi-synthesis. Copyright

Full text of DOI:10.1002/anie.201302065

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DOI: 10.1002/anie.201302065
Protein Semi-Synthesis
Expressed Protein Ligation at Methionine: N-Terminal Attachment of
Homocysteine, Ligation, and Masking**
Tomohiro Tanaka, Anne M. Wagner, John B. Warner, Yanxin J. Wang, and E. James Petersson*
The synthesis of chemically modified proteins is a valuable
tool for the study of their function.[1] One of the most enabling
protein synthesis technologies is native chemical ligation
(NCL), in which a peptide thioester is reacted with a peptide
bearing an N-terminal Cys to form a native amide bond.[2] In
many cases, one of these two fragments can be expressed in E.
coli and then ligated to a smaller synthetic peptide so that
a large semi-synthetic protein can be made with a minimum of
peptide synthesis. This sort of expressed protein ligation
(EPL) can be implemented in two ways.[3] For a synthetic C-
terminus, the N-terminal region can be expressed as a fusion
to an intein and then converted to a thioester for ligation.
Likewise, a protein can be expressed with an N-terminal Cys
and ligated to a synthetic thioester. EPL is an extremely
efficient way of making large quantities of synthetic proteins.
However, there are limitations to the method, most notably,
the need for Cys at the site of ligation.
Since many proteins do not have Cys residues at
convenient locations or lack Cys entirely, several research
groups have developed methods for “erasing” the ligation
site. The most common strategies are alkylation of Cys to
make Lys or Glu analogs and desulfurization to convert Cys
to Ala.[4] Danishefsky and others have developed strategies
using synthetic Cys analogs that are converted to other amino
acids such as Leu, Phe, Val, Thr, and Lys by desulfurization.[5]
Dawson and Muir have used nitrobenzyl Cys surrogates that
can be removed by photolysis after ligation.[6] Raines and
Bertozzi have demonstrated “traceless” ligations based on
Staudinger reductions and Bode has developed a novel
ligation using ketoacid/alkoxyamine partners.[7] Homocys-
teine (Hcs) has been used for ligation and then converted to
Met by selective methylation.[8] While chemically elegant,
most of these strategies are still limited in that they require
the ligation handles to be incorporated synthetically. There-
fore, these handles can be placed in a synthetic peptide to be
ligated to an intein-derived thioester, but cannot be applied to
an expressed C-terminal fragment for ligation to a synthetic
thioester.
One way of functionalizing protein N-termini makes use
of the aminoacyl transferase (AaT) enzyme from E. coli,
which transfers Phe, Leu, or Met from an aminoacyl tRNA
(Zaa-tRNA) to the N-terminus of a protein.[9,10] Our group
has recently shown that AaT does not require full-length
tRNAs, and can use aminoacyl adenosine (Zaa-A) substrates
to transfer a variety of amino acids.[11] Previous work from
Tirrell and Sisido demonstrated that AaT could transfer
unnatural amino acids from Zaa-tRNAs produced either
through semi-synthesis or by the activity of a modified
aminoacyl tRNA synthetase (RS).[12] These two strategies are
complementary. Our Zaa-A donor substrates are easily
synthesized and allow us to explore the inherent substrate
specificity of AaT without the necessity of identifying a novel
RS. Once a useful substrate is identified, higher yields can be
achieved with the catalytic regeneration of Zaa-tRNA by an
appropriate RS. We wished to determine whether AaT could
be used to functionalize proteins with erasable Cys analogs
for ligation. Varshavsky and others have shown that AaT
recognizes N-terminal Arg or Lys residues regardless of the
adjacent sequence.[10,13] Therefore, we expected that any
transferable analogs we identified would be applicable to
a wide variety of protein targets. Here, we show that AaT can
deliver disulfide-protected Hcs to the N-terminus of a protein
under mild conditions. After reduction, Hcs can be used in
a ligation reaction and then converted to Met by alkylation
(Figure 1). We demonstrate the utility of this approach by
[*] Dr. T. Tanaka, A. M. Wagner, J. B. Warner, Y. J. Wang,
Prof. E. J. Petersson
Department of Chemistry, University of Pennsylvania
231 South 34th Street, Philadelphia, PA 19104-6323 (USA)
E-mail: ejpetersson@sas.upenn.edu
Homepage: http://ejpweb1.chem.upenn.edu/
[**] This work was supported by funding from the University of
Pennsylvania and the Searle Scholars Program (10-SSP-214 to
E.J.P.). T.T. was supported by a fellowship from the Japan Society for
the Promotion of Science (JSPS). We thank Rakesh Kohli for
assistance with HRMS (NIH RR-023444) and MALDI-MS (sup-
ported by NSF MRI-0820996).
Figure 1. Expressed protein ligation at methionine. An expressed
protein bearing an N-terminal Arg or Lys (green) is functionalized with
a protected Hcs analog from an aminoacyl adenosine donor, under
catalysis by AaT transferase. A synthetic peptide thioester (red) is
ligated to the modified protein under reducing conditions that allow
deprotection of Hcs. After ligation, Hcs is converted to Met.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201302065.
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Scheme 2. Amino acid transfer to reporter peptide. Reagents and
conditions: a) 1 mm Zaa-A, 0.1 mgmLÀ1 AaT; or b) 1 mm Zaa,
2 mgmLÀ1 E. coli total tRNA, 0.1 mgmLÀ1 Met*RS, 0.1 mgmLÀ1 AaT;
with 100 mm LysAlaAcm in 50 mm HEPES, 150 mm KCl, 10 mm MgCl2,
pH 8.0, 378C, 4 h. Yields determined by HPLC analysis.
Scheme 1. Synthesis of protected homocysteine donor molecules.
Reagents and conditions: a) (Boc)2O, DIPEA, THF; b) RSH, NaOH, I2,
THF/H2O (1:1); c) ClCH2CN, DIPEA, THF; d) (DMT)-A, DIPEA,
NBu4Ac, THF; e) TFA, TIPSH, THF. 2a, 3a, 4a, 5a R=Me; 2b, 3b,
4b, 5b R=iPr; 2c, 3c, 4c, 5c R=tBu; 2d, 3d, 4d, 5d R=Ph. Yields
reported in Supporting Information. Boc=tert-butyloxycarbonyl,
DIPEA=N,N-diisopropylethylamine, THF=tetrahydrofuran, (DMT)-
A=5’-O-dimethoxytrityl adenosine, NBu4Ac=N,N,N,N-tetrabutylam-
monium acetate, TFA=trifluoroacetic acid, TIPSH=triisopropylsilane.
yield. While this was lower than Met, it established that we
could transfer disulfide-protected analogs without substantial
side reactions. Hcs donors 5a–d were tested in a similar
fashion. Hcm was transferred efficiently from 5a, with
somewhat lower yields for Hcp and Hcb. No transfer of Hcf
was observed from donor 5d. In order to maintain the
disulfide-protected analogs during the transfer reaction, we
eliminated b-mercaptoethanol (BME) from the AaT purifi-
cation process and the reaction buffer. This increased the
yields of HcmLysAlaAcm (6) to 37%. Once Hcm was
identified as an optimal substrate, we assessed the possibility
of generating Hcm-tRNA in situ using an RS. Free Hcm was
synthesized by TFA deprotection of Boc-Hcm. Wild-type E.
coli MetRS and an L13G mutant (Met*RS) previously used by
the Tirrell group were tested with Hcm, ATP, and E. coli total
tRNA.[16] Met*RS was more active than MetRS, giving full
transfer at ꢀ 0.1 mgmLÀ1 loading. This is consistent with
Met*RS structural analysis, where longer sidechains are
accommodated by the expanded active site.[16]
synthesizing a-synuclein, a protein whose aggregation con-
tributes to Parkinsonꢀs disease pathology.[14]
Previous studies had shown that Cys-tRNA is not a sub-
strate for AaT and our own preliminary experiments showed
that Hcs was not transferred (data not shown).[13,15] Therefore,
we prepared Hcs analogs masked as aliphatic or aromatic
disulfides in order to mimic the endogenous AaT substrates
Met, Leu, and Phe. Disulfide protecting groups (MeS, iPrS,
tBuS, and PhS) were used so that they could be removed
in situ under the reducing conditions of the subsequent NCL
reaction.
AaT donor molecules were synthesized using a simple
protocol beginning with Boc protection of oxidized Hcs dimer
to give 1, followed by disulfide exchange to form protected
Hcs analogs 2a–d (Scheme 1) These were activated as
cyanomethyl esters 3a–d, allowing for acylation of the 2’
and 3’ hydroxy groups of 5’-O-dimethoxytrityl-adenosine,
(DMT)-A, without amidation of its unprotected exocyclic
amine. TFA treatment cleaved both the DMT and the Boc
groups to give Zaa-A donor molecules 5a–d. Overall yields of
the DMT-protected adenosyl analogs (4a–d) were ca. 40%
from (Hcs)2. However, several byproducts of the TFA
deprotection reaction were observed, requiring HPLC purifi-
cation of 5a–d.
We then took HcmLysAlaAcm (6) on to a model ligation
reaction with Ac-MetAspValPhe-SR (7) (Scheme 3). HPLC
and MALDI MS analyses showed that disulfide cleavage was
Once the Zaa-A donor molecules were purified, they
were tested in transfer reactions to a reporter peptide
LysAlaAcm, where Acm is 7-aminocoumarin. We compared
yields of ZaaLysAlaAcm by HPLC analysis after 4 h reac-
tions at pH 8.0 at 378C (Scheme 2). Under these conditions,
transfer of Phe is quantitative and Met proceeds in 75% yield.
We first tested the transfer of disulfide-protected compounds
using thiomethyl Cys (Csm), a Met isostere, prepared through
a route similar to Scheme 1. Csm was transferred in 21%
Scheme 3. Ligation and alkylation of Hcs-functionalized peptide.
Reagents and conditions: a) 1.0 mm 6, 3.3 mm 7, 100 mm sodium
phosphate dibasic, 20 mm TCEP, 0.5% PhSH, 0.5% BnSH, pH 7.2,
room temperature, 24 h, 100%; b) 50 mm MeI, 100 mm sodium
bicarbonate, pH 8.6, room temperature, 5 min, 89%.
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completed almost immediately and that the subsequent
ligation reached 100% completion after 24 h. After ligation,
the product peptide (8a) was treated with 1000 equiv MeI at
pH 8.6 to form 8b. After 5 min, 89% conversion of Hcs5 to
Met5 was observed by HPLC and MALDI MS analyses
(Supporting Information). Unreacted 8a (11%) could be
removed after disulfide formation. Oxidation of Met1, was
observed (4%), but no undesired alkylation of Met or Lys was
seen. This is consistent with previous uses of Hcs as a ligation
handle, where subsequent alkylation is surprisingly selective,
given that the pKa of Hcs (8.9) is close to the pKa of Lys
(9.5).[17]
After successful completion of the model peptide ligation,
we wished to test the Hcs transfer/ligation strategy with a full-
sized protein. One protein that we have previously studied
which has the requisite MetLys motif is a-synuclein (aS).
Several recent studies have shown that the N-terminal
sequence of aS can have important consequences for its
folding and self-association.[18] However, these studies pro-
foundly disagree on what those consequences are. Therefore,
the preparation of N-terminally modified aS could be useful
to addressing these questions and serve as a valuable
demonstration of our method.
The aS NCL protocol is shown in Figure 2. We chose
Met5Lys6 as the point of disconnection and prepared an aS6–140
plasmid with an N-terminal His10 tag and a Factor Xa
proteolysis site. The precursor protein, HisTag-aS6–140 (9), was
isolated by Ni-affinity chromatography and cleaved with
Factor Xa to give aS6–140 (10). We incubated 10 with AaT,
Met*RS, tRNA, Hcm, and ATP. Transfer of Hcm was
complete after 1 h, within the limits of quantitation by
MALDI MS (Supporting Information). Complete transfer is
extremely valuable for large proteins, since separation of an
Hcm-modified protein such as 11 from unmodified 10 is not
generally feasible. Modified aS 11 was isolated from AaT,
Met*RS, and tRNA by boiling and FPLC purification. Then
11 was incubated with thioester AcMcmMetAspValPhe-SR
(S13), which corresponds to aS1–4 labeled at the N-terminus
with 7-methoxycoumarinylalanine (Mcm). Overnight incuba-
tion at 378C gave 12a in high yield from 11. No unligated
starting materials were observed, though some oxidized 12a
was observed (Supporting Information). Purified 12a was
quantitatively converted to 13a by treatment with aqueous
MeI, as determined by analyses using Ellmanꢀs reagent and
MALDI MS (Supporting Information). In general, we expect
that the NCL reaction will not be 100% efficient, but we do
not view this as a severe limitation, provided that the NCL
product can be purified away from the starting materials and
methylation is quantitative and selective.
A similar ligation was carried out with Ac-MetAspVal-
Phe-SR (7), yielding N-Ac-aS after methylation (13b). The
ligation product 13b was subjected to trypsin digest and
MALDI MS analysis to confirm that methylation occurred
with high yield and few side reactions. Observation of the Ac-
MetAspValPheMetLys fragment (Ac-aS1–6) with only trace
Ac-MetAspValPheHcsLys contamination confirmed that
methylation proceeded in > 95% yield within the limitations
of quantitation by MS. Furthermore, MALDI MS analysis of
the rest of the tryptic fragments demonstrated that there were
no substantial alkylation side reactions, even on His50 in the
aS46–58 fragment (Supporting Information). Such selectivity is
rewarding, but probably not completely general; for any
novel protein, similar analysis by digestion and MS should be
performed. It should also be noted that protein targets
containing unprotected Cys residues would also be methy-
lated on the free thiols. However, more conventional NCL
strategies using ligation at Cys are probably viable in those
cases.
Application of our system to targets other than aS may
require mild denaturation of the protein in order to access the
N-terminal amino acids. Therefore, we have tested AaT
activity in the presence of low concentrations of denaturants
and detergents. We find that AaT activity has a Gdn·HCl IC50
of 0.4m and that full activity could be maintained in 5% v/v
Triton X-100. While the N-termini of some proteins may be
buried in important structural interactions, these concentra-
tions should allow one to partially unfold many proteins to
access the terminus for Hcm transfer and ligation.
While our experiments begin to remove the sequence
limitations for EPL, they certainly do not eliminate them
entirely. Essentially, one can now use XxxMetArg or
XxxMetLys as a disconnection point in a retrosynthetic
analysis of a potential protein target. The utility of our
approach depends on how frequently this motif occurs. To
address this, we analyzed the protein sequences in the PDB
and found that out of the 60325 proteins surveyed, 5405
possible N-terminal ligation sites (< 40 amino acids from the
N-terminus) were identified in 5156 unique proteins. These
numbers increased to 31259 ligation sites and 20701 proteins
when we considered proteins with the ligation motif in the
center of the protein where one might use multiple ligations
to synthesize the full-length protein. We chose to restrict our
Figure 2. a-Synuclein model ligation. Functionalization of aS6–140 by
a) cleavage of His tag at Factor Xa site, b) attachment of Hcm by AaT-
catalyzed modification, c) ligation of an N-terminal thioester peptide
(7 or S13), and d) conversion of Hcs to Met by methylation. 12a, 13a
Xxx=AcMcm; 12b, 13b Xxx=Ac. Top right: Gel image showing
product of each step, WT indicates full-length aS. Middle right:
MALDI MS analysis of full length Ac-aS before (12b) and after (13b)
methylation. Bottom right: Trypsinized fragment corresponding to Ac-
aS1–6 (812.4 m/z) confirms successful methylation. Asterisk indicates
the expected mass of unmethylated Ac-aS1–6Hcs5.
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Received: March 12, 2013
Published online: April 29, 2013
Keywords: chemoenzymatic reactions · chemoselectivity ·
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ligation reactions · protein synthesis · a-synuclein
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