DOI: 10.1002/anie.201100840
Protein Chemistry
One-Pot Dual-Labeling of a Protein by Two Chemoselective
Reactions**
Long Yi, Hongyan Sun, Aymelt Itzen, Gemma Triola, Herbert Waldmann, Roger S. Goody,* and
Yao-Wen Wu*
Multicolor labeling is a valuable technique for the character-
ization of proteins with respect to their structure, folding, and
interactions both as single molecules and in cellular inves-
tigations. The key technique for such studies is based on
fluorescence resonance energy transfer (FRET).[1] FRET
applications require the attachment of donor (D) and
acceptor (A) molecules to specific sites of a given protein or
proteins. Such labeling is typically achieved through con-
jugation at cysteine residues or amino groups or by genetic
fusion to different fluorescent proteins.[2–5] Recent advance-
ments in chemical methods have substantially expanded the
tools that are available for site-specific modification of
proteins.[6] However, site-specific incorporation of multiple
fluorophores into a single protein remains a considerable
challenge. Dual labeling of a single protein has been achieved
using multistep reactions. For example, sortases with different
substrate specificity were used for site-specific C- and N-
terminal labeling of a single protein.[7] Muir and Cotton
reported a method for producing a dual-labeled protein
through a multistep expressed protein ligation approach.[8]
Recently, Yang and Yang used a three-step strategy based on
split inteins for site-specific two-color protein labeling.[9]
Herein, we report a facile and efficient method for dual-
labeling of proteins based on chemoselective reactions.
Frequently used chemoselective reactions include native
chemical ligation (NCL), Staudinger ligation, Huisgen 1,3-
dipolar cycloaddition (click chemistry), oxime ligation, strain-
promoted cycloaddition, and Diels–Alder ligation.[10] We
reasoned that by employing two chemoselective reactions
for protein labeling, it should be possible to obtain two-color
labeled proteins in a one-pot reaction in a straightforward
fashion.
Recently, we reported a method for intein-mediated
incorporation of a (bis)oxyamine moiety into the C terminus
of proteins, making them amenable to efficient conjugation
with a keto fluorophore under mild conditions.[11] For N-
terminal labeling, a protein containing an N-terminal cysteine
can undergo NCL with thioester probes.[12] The exposure of an
N-terminal cysteine can be achieved by TEV (tobacco etch
virus) protease cleavage. Hence, we speculated that both
NCL and oxime ligation could be employed for one-pot two-
color labeling of a given protein.[13] Herein we present a
strategy for constructing a dual-labeled Rab7 GTPase in a
one-pot reaction and illustrate the use of the method for
studying protein refolding and protein–protein interactions.
To generate Rab7 with an N-terminal cysteine, we fused a
peptide sequence that provides
a TEV cleavage site
(ENLYFQ:C; the dotted line indicates the cleavage site) to
the N terminus of the Rab7D3 protein. Rab7D3 fused N-
terminally to an engineered Mxe GyrA intein domain can
undergo initial N!S acyl transfer and be subsequently
cleaved by thiol reagents (such as 2-mercapoethanesulfonic
acid) by an intermolecular transthioesterification reaction,
releasing a a-thioester-tagged protein.[14] Subsequently, the
Rab7D3-thioester (10 mgmLꢀ1, 400 mm) was treated with
500 mm (bis)oxyamine at pH 7.5 to produce the oxyamine
protein derivative. TEV protease was then added to cleave
the N-terminal protection sequence (Scheme 1). This led to a
doubly functionalized Rab7 protein with an N-terminal
cysteine and a C-terminal oxyamine, N-Cys-Rab7D3-ONH2,
for chemoselective reactions.
Herein, we chose coumarin thioester (quantum yield of
0.27) as FRET donor and keto fluorescein (quantum yield of
0.97) as FRET acceptor, both of which can be easily prepared
from commercially available reagents (for details, see the
Supporting Information). R0 for the coumarin and fluorescein
pair is 47 ꢀ. Our first goal was to achieve quantitative
conversion for both reactions. The NCL reaction for N-
terminal modification was not complete after incubation for
three days with 2-mercaptoethanesulfonate (MESNA) as a
thiol cofactor (data not shown). The addition of 100–200 mm
(4-carboxymethyl)thiophenol (MPAA) significantly acceler-
ated the reaction,[15] and the reaction of N-Cys-Rab7D3-
ONH2 (1 mgmLꢀ1, 43 mm) with 0.7 mm coumarin thioester
was complete in 2–3 h at pH 7.0 at room temperature or 12 h
on ice with quantitative conversion (see the Supporting
Information). The C-terminal oxime ligation of N-Cys-
[*] L. Yi,[+] Dr. A. Itzen, Prof. R. S. Goody, Dr. Y. W. Wu
Department of Physical Biochemistry
Max-Planck-Institut fꢀr molekulare Physiologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
E-mail: yaowen.wu@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
Technische Universitꢁt Dortmund
44227 Dortmund (Germany)
[+] These authors contributed equally to this work.
[**] We thank Nathalie Bleimling for excellent technical help. We thank
Matthias Machner for the gift of the LidA plasmid. L.Y. acknowl-
edges a Ph.D. fellowship from the International Max Planck
Research School (IMPRS) in Chemical Biology. H.S. thanks the
Humboldt Foundation for a Humboldt fellowship.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2011, 50, 8287 –8290
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
8287