A highly efficient strategy for modification of proteins at the C terminus

Article abstract of DOI:10.1002/anie.201003834

A new linker: In a facile, chemoselective, and potentially general method for protein modification at the C terminus oxyamino-modified proteins obtained from protein thioesters react rapidly with ketones under mild conditions. This strategy was used for fluorescence labeling, for example with modified coumarin and fluorescein (see scheme). Copyright

Full text of DOI:10.1002/anie.201003834

Angewandte
Chemie
DOI: 10.1002/anie.201003834
Protein Chemistry
A Highly Efficient Strategy for Modification of Proteins at the
C Terminus**
Long Yi, Hongyan Sun, Yao-Wen Wu, Gemma Triola, Herbert Waldmann,* and
Roger S. Goody*
Site-specific protein modification can facilitate the character-
ization of proteins with respect to their structure, folding, and
interaction with other proteins both in biochemical and in
cellular investigations. Although many chemical reactions are
applicable in principle, methods for the site-specific modifi-
cation of proteins remain in high demand, and there is a
requirement for readily available ligation reagents and mild
reaction conditions.[1] Oxime-based reactions have found
wide application in the conjugation of biomolecules on
account of the absence of oxyamino groups in proteins and
their orthogonal reactivity with ketones to give stable
oximes.[2–5] The oxyamine–ketone bioorthogonal reaction
has been exploited in protein modification mainly by means
of incorporating ketone groups into proteins by various
chemical,[6] enzymatic,[7] and molecular biological[8] methods.
To expand the application of this efficient methodology to
protein ligation, we planned the development of simple and
general methods to incorporate oxyamino groups into pro-
teins. In an earlier approach to the use of auxiliary groups in
native chemical ligation, it was shown that the nitrogen atom
of oxyamines can react with a thioester group intramolecu-
larly.[9] On the other hand, aminolysis of peptide and protein
thioesters has been successfully reported in some studies.[10]
We reasoned that in theory, highly nucleophilic oxyamines
could also react with a thioester moiety in a protein
intermolecularly. If this reaction would be performed with a
linker carrying two oxyamino groups, then one oxyamine
could form a hydroxamic acid bond to the protein and the
second would still be available for a subsequent ligation
reaction with the selectively functionalized protein. Herein,
we describe how a bis(oxyamine) molecule was first incorpo-
rated at protein C termini to produce oxyamino-modified
proteins, and how the subsequent efficient and specific
reaction of the second oxyamino group with ketones was
achieved to modify proteins site-specifically under mild
conditions.
The unique position and chemistry of protein C termini
has stimulated efforts to target this location for site-selective
protein modification. In one approach, a protein tag is
appended to a target protein, and an enzymatic reaction is
used to covalently introduce a C-terminal modification onto a
protein.[11–13] However, the protein tag is still retained in the
labeled protein, which may interfere with protein function.
An intein-based protein-cleavage reaction has generated very
useful approaches to the C-terminal modification of pro-
teins,[14] mainly based on thioester-mediated ligation chemis-
try.[15] A limitation of this method is that it generally leads to
introduction of a cysteine residue into the target protein,
regardless of whether this corresponds to the native structure
or not. In addition, the thioester reaction frequently proceeds
at relatively slow ligation rates and requires a relatively high
protein concentration. Recently, it was reported that the C-
terminal carboxylate can also be transformed into a thioacid,
followed by C-terminal modification of the protein by means
of thioacid/azide amidation in the presence of 6m guanidine
hydrochloride (Gdn-HCl) containing 3 mm 2,6-lutidine.[16]
The modified conditions are too harsh to maintain the
proper folding of a protein and the thioacid group is prone
to hydrolysis. In contrast to these reports, we could introduce
an oxyamino group into the C terminus of protein in
phosphate buffer (pH 7.5), and the modified protein can
subsequently undergo fast and chemoselective oxime ligation
on ice.
The synthesis of 1,2-bis(oxyamino)ethane (1) started from
the commercially available and inexpensive reagents N-
hydroxyphthalimide and 1,2-dibromoethane (Figure 1).[17]
Bis(oxyamine) 1 was obtained in a convenient manner
without chromatography (> 20% yield over two steps; for
details see the Supporting Information). The facile and
economic synthesis of 1 is important for the wide use of the
method reported herein. Rab1bD3-thioester (Ras-related
GTPase) generated through intein-mediated partial protein
splicing was chosen as the model protein. In order to
introduce an oxyamino group into the protein, the reaction
of Rab1bD3-thioester and 1 was performed on ice for 4 h
(quantitative conversion). As shown in Figure 1, the bis-
[*] L. Yi,[+] Dr. Y.-W. Wu, Prof. R. S. Goody
Department of Physical Biochemistry
Max-Planck-Institut fꢀr molekulare Physiologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
Fax: (+49)231-133-2399
E-mail: roger.goody@mpi-dortmund.mpg.de
L. Yi,[+] Dr. H. Sun,[+] Dr. G. Triola, Prof. H. Waldmann
Department of Chemical Biology
Max-Planck-Institut fꢀr molekulare Physiologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
and
Chemische Biologie, Fachbereich Chemie
Universitꢁt Dortmund, 44227 Dortmund (Germany)
Fax: (+49)231-133-2499
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
[+] These authors contributed equally to this work.
[**] We thank Nathalie Bleimling for the gift of Rab1bD3, Christiane
Theiss for the cloning of Rab7D7. L.Y. acknowledges a PhD
fellowship from the International Max Planck Research School
(IMPRS) in Chemical Biology. H.S. thanks the Humboldt Founda-
tion for a Humboldt fellowship.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201003834.
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Figure 1. a) Synthesis of bis(oxyamine) 1 and the generation of Rab-
ONH2. b,c) ESI mass spectra of Rab1bD3 and Rab1bD3-ONH2,
respectively.
(oxyamine) reacted directly with the thioester group to form a
stable O-alkyl hydroxamic acid bond. ESI-MS analysis
revealed that the main protein peak had
a mass of
22329 Da (expected mass 22330 Da based on Rab1bD3),
implying that most of the Rab1bD3 protein was modified with
the oxyamino moiety. It should be noted that there is no sign
of bis(oxyamino)-bridged proteins (see SDS-PAGE in
Figure 2). There was also no detectable protein hydrolysis
during this reaction. Unreacted 1 was removed from the
Rab1bD3-ONH2 by simple dialysis against sodium phosphate
buffer (pH 7.5).
Figure 2. a) Fluorescence labeling at the C terminus by oxyamine–
ketone ligation. b) Fluorescence (left) and Coomassie staining (right)
images of a gel loaded with Rab1bD3-ONH2 labeled with keto-
fluorescein 3 (M: marker of 97, 66, 45, 30, 20.1, 14.4 kDa; lane 1:
Rab1bD3-ONH2; lane 2: Rab1bD3-thioester and keto-fluorescein for
30 min incubation on ice; lanes 3–5: reaction between Rab1bD3-ONH2
and keto-fluorescein for 30 min, 1 h, 2 h on ice, respectively). c) ESI
mass spectrum of the Rab1bD3–fluorescein ligation product.
The reactivity of the oxyamino group on the protein was
first tested by reaction with acetone. After simple incubation
of Rab1bD3-ONH2 (1 mgmLÀ1) and acetone (10 mm) at
pH 5.5 on ice, the reaction was finished within 20 min as
shown by ESI-MS (Figure S3 in the Supporting Information),
indicating that the reaction was rapid and generated a protein
carrying a single acetone modification. In order to achieve
fluorescent labeling of proteins, we first chose coumarin, since
this fluorophore is highly soluble in water, has an excellent
quantum yield, and is easy to synthesize (Figure 2).[18] On
incubation of keto-coumarin 2 and Rab1bD3-ONH2 on ice for
4 h, full conversion of the oxyamine to an oxime was
achieved, as shown by ESI-MS. Excitation/emission scans of
a solution of Rab1bD3–coumarin revealed that, as expected
from the reporter group used, the fluorescence spectrum has
an excitation maximum at 332 nm (data not shown) and an
emission maximum at 412 nm (Figure S4 in the Supporting
Information). In order to examine the efficiency of the
fluorescent labeling by SDS-PAGE, keto-fluorescein 3 was
synthesized and incubated with Rab1bD3-ONH2 on ice for
various reaction times. The fluorescence gel image (Fig-
ure 2b) showed that the reaction was rapid and complete
within 1 h on ice. Rab1bD3-thioester did not show any
labeling with keto-fluorescein 3, further confirming that the
reaction was bioorthogonal. The labeling reaction appeared
to be highly efficient, since none of the original Rab1bD3-
ONH2 was detected by ESI-MS (Figure 2c).
To further validate this ligation method as a general
method for C-terminal modification of proteins, it was also
applied to enhanced yellow fluorescent protein (EYFP). As
for Rab1b, the EYFP thioester was generated from an intein
fusion protein, which was further modified with the oxyamino
group by direct incubation of protein and bis(oxyamine) 1 in
buffer. The obtained EYFP-ONH2 was then incubated with
keto-fluorescein 3 for different times on ice (Figure S6 in the
Supporting Information). The fluorescence intensity of the
band corresponding to the product increased with time, and
the reaction was essentially complete after 30 min. Formation
of the product with the expected mass was shown by ESI-MS.
In order to determine whether proteins still retain their
activity after fluorescent labeling, we chose another Rab
GTPase, Rab7, which binds to Rab escort protein (REP-1)
with nanomolar affinity.[19] Fluorescence labeling of Rab7D2
was performed as for Rab1bD3 with keto-coumarin 2. In the
ESI mass spectrum the protein peaks have masses corre-
sponding to Rab7D2-ONH2 and Rab7-coumarin, 23273 and
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23458 Da, respectively, consistent with the expected values of
23275 and 23459 Da (Table S1 in the Supporting Informa-
tion). Rab7D2-coumarin and REP-1 were incubated on ice for
1 h, separated by gel filtration, and analyzed by SDS-PAGE.
As shown in Figure S8 (Supporting Information), it is clear
that Rab7-coumarin and REP-1 form a complex, implying
that the modification of coumarin at the C terminus did not
significantly perturb its interaction with REP-1.[20]
To assess whether this method can be used for studying
protein–protein interactions, we attached an environmentally
sensitive fluorescence probe, keto-dansyl 4, to the protein
C terminus by oxime ligation. When the semisynthetic
Rab7D7-dansyl was titrated with increasing concentrations
of REP-1, a dose-dependent and saturatable increase of the
fluorescence emission signal at 500 nm was observed upon
excitation at 340 nm (Figure 3). Titration data could be fitted
Figure 4. a) Schematic representation of the generation of a dual-color
protein showing FRET between fluorescein and mcherry. b) Emission
spectra of mcherry-Ypt7D3-fluorescein showing a FRET effect, while the
denatured protein just shows the emission band of fluorescein.
within the mcherry-Ypt7D3-fluorescein molecule, since a
strong fluorescent signal at 610 nm could be observed upon
excitation at 490 nm (excitation wavelength for fluorescein).
Compared with mcherry-Ypt7D3-ONH2 at the same concen-
tration and excited at the same wavelength, this represents a
roughly sixfold increase in signal intensity due to FRET. In
contrast, denatured mcherry-Ypt7D3-fluorescein displayed
only a strong signal at the emission wavelength 510 nm,
suggesting that the FRET disappeared because mcherry
unfolded. FRET could provide a sensitive signal to test the
folding state of the construct under the ligation conditions
(i.e. at pH 5.5). Thus, storing mcherry-Ypt7D3-fluorescein in
the ligation buffer for one week on ice did not lead to
significant change in the fluorescence spectra (data not
shown), further suggesting that the ligation conditions are
mild enough for general protein modification, although this
remains to be tested further in individual cases.
Oxime ligations normally proceed at modest reaction
rates in acidic solution.[3b] The rate of oxime ligation in this
study seems to be significantly faster than those reported
when a keto group was incorporated into proteins.[9,23] For the
five proteins in this study, all ligation reactions at pH 5.5 were
complete within 4 hours on simple incubation of protein-
ONH2 and the ketone fluorophore on ice. Further experi-
ments showed that the oxime reaction could also be achieved
quantitatively at pH 6.5 (Figure S11 in the Supporting Infor-
mation). Surprisingly, the ligation could be accomplished
efficiently at pH 7.0 within 2 days by incubating 1 mgmLÀ1
Rab1D3-ONH2 and 1 mm keto-fluorescein 3 on ice (Figure 5
and Figure S12 in the Supporting Information). Aniline can
catalyze the oxime ligation in neutral buffer,[5a] and in our case
the ligation rate can be further improved (by about fivefold)
in the presence of 100 mm aniline at pH 7.0 (Figure 5).
We found that the oxime bond was stable enough at
neutral pH to allow typical applications in biochemical
Figure 3. Emission spectra (excitation at 340 nm) of Rab7D7-dansyl
(140 nm) upon addition of various concentrations of REP-1. Dotted
line: blank, background. The gray lines represent the spectra obtained
with various concentrations of REP-1 (0–861 nm, see the Supporting
Information for details). The fluorescent signal increases with increas-
ing concentration of REP-1.
using a quadratic equation describing the binding curve and
were consistent with 1:1 stoichiometry (Figure S9 in the
Supporting Information). The Kd value obtained (2.6 nm) is
close to that determined previously using mant-GDP-bound
Rab7 (1.0 nm).[21] Our results demonstrate that the oxyamine–
ketone ligation offers an advantageous and novel approach
for the site-selective functionalization and labeling of proteins
for studies of protein–protein interactions.
In a further application of the method, we applied it to the
generation of a doubly labeled Rab. Dual-color protein
labeling is a valuable tool for fluorescence resonance energy
transfer (FRET) studies of dynamic biomolecular dynamics
and associations.[22] We expressed the protein mcherry-
Ypt7D3-intein, since mcherry is a FRET acceptor for
fluorescein. After performing the labeling reaction of
mcherry-Ypt7D3-ONH2 with keto-fluorescein 3 on ice for
2 h, we removed excess keto-fluorescein using NAP-5 col-
umns (GE Healthcare). Fluorescence imaging of the gel
shows dual-color labeling of the protein (Figure S10 in the
Supporting Information). Figure 4 shows that FREToccurred
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Keywords: fluorescent probes · immobilization · oxime ligation ·
.
protein modification · site-specific modification
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Received: June 23, 2010
Published online: October 28, 2010
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