The binding of fluorophores to proteins depends on the cellular environment

Article abstract of DOI:10.1002/anie.201007626

Lighten up! To investigate intracellular binders of myotube-specific probes, a thiol-reactive derivative, CDy2, was prepared. In live cells, the derivative selectively localizes in mitochondria and covalently labels its binding partners. Copyright

Full text of DOI:10.1002/anie.201007626

DOI: 10.1002/anie.201007626
Fluorophore-Binding Proteins
The Binding of Fluorophores to Proteins Depends on the Cellular
Environment**
Yun Kyung Kim, Jun-Seok Lee, Xuezhi Bi, Hyung-Ho Ha, Shin Hui Ng, Young-hoon Ahn,
Jae-Jung Lee, Bridget K. Wagner, Paul A. Clemons, and Young-Tae Chang*
Fluorescent small molecules are extensively used for live-cell
imaging, but mainly in the context of labeling conjugates for
other protein-binding motifs, such as antibodies.[1] As most
fluorescent molecules are flat and hydrophobic, it has
generally been believed that these fluorophores may bind to
many hydrophobic proteins in cells without any specificity.[2]
This conventional wisdom, however, has not been tested
systematically because of the lack of sufficiently diverse dye
sources. Recently, we developed a diversity-oriented fluores-
cence library approach (DOFLA) to use fluorescent dyes to
distinguish directly cellular components such as GTP,[3]
DNA,[4] RNA,[5] heparin,[6] and organelles.[7] In this system,
the diverse structural motifs of each dye molecule in the
library endowed target selectivity. From these results, we
envisioned that sufficient structural modifications of fluoro-
phore scaffold could lead us to develop probes that label
specific proteins from whole proteomes.[8] As an extension of
our recent finding of a fluorescein derivative labeling
glutathione s-transferase,[9] here we report a rosamine deriv-
ative that labels tubulin in vitro and a mitochondrial protein
in live cells.
Previously a fluorescent small molecule capable of
detecting differentiated myotubes was discovered from a
mitochondria-targeted rosamine library.[10–12] The hallmark of
muscle differentiation is the fusion of mononucleated myo-
blasts to multinucleated myotubes.[13] During murine C2C12
myogenesis, the fluorescence intensity of one rosamine
compound, E26, increased significantly. This myotube selec-
tivity may be achieved by binding to one of the differentiation
markers expressed more highly in myotubes; alternatively,
the probe may detect other physiological changes after
differentiation. When subjected to further investigation,
unfortunately, E26 showed photo-instability under strong
and continuous light irradiation (see Figure S1 in the Sup-
porting Information). The compounds in the rosamine library
were retested under extended exposure to light and we
selected two compounds (I25 and I31; Figure 1) based on
their high photostability and fluorescence response after
differentiation (fluorescence response: I25: (2.4 Æ 0.2)-fold,
I31: (3.0 Æ 0.5)-fold, N = 3; Figure S2).
[*] Dr. Y. K. Kim, Dr. J.-S. Lee, Dr. H.-H. Ha, Prof. Dr. Y.-T. Chang
Department of Chemistry, National University of Singapore
Singapore 11754 (Singapore)
In order to identify protein binders of these compounds,
affinity matrices were prepared based on careful studies of the
structure–activity relationships (Figure 2a). The affinity pull-
Fax: (+65)6779-6591
E-mail: chmcyt@nus.edu.sg
Homepage: http://ytchang.science.nus.edu.sg
Dr. Y. K. Kim, Dr. J.-S. Lee
Korea Institute of Science and Technology (KIST)
Life/Health Division, Integrated Omics Center, Seoul (Korea)
Dr. X. Bi, S. H. Ng, Dr. J.-J. Lee, Prof. Dr. Y.-T. Chang
Laboratory of Bioimaging Probe Development
Singapore Bioimaging Consortium, A*STAR, Biopolis
Singapore
Dr. H.-H. Ha, Prof. Dr. Y.-T. Chang
NUS MedChem Program of the Office of Life Sciences
National University of Singapore, Singapore
Dr. Y.-h. Ahn
Department of Pharmacology, Johns Hopkins University
Baltimore, MD (USA)
Dr. B. K. Wagner, Dr. P. A. Clemons
Chemical Biology Program
Broad Institute of Harvard and MIT, Cambridge, MA (USA)
[**] This work was supported by intramural funding from A*STAR
Biomedical Research Council and a National University of Singa-
pore Young Investigator Award (R-143-000-353-123). We gratefully
acknowledge the U.S. National Institutes of Health for financial
support (P20-GM072029 to Y.-T.C. and P.A.C.; P.A.C. is also
supported in part by RL1-HG004671). J.-S.L. is grateful for the
generous support of the POSCO TJ Park Foundation (TJ Park
Bessemer Science Fellowship).
Figure 1. Probes for myogenic differentiation. a) Screen of rosamine
library. Myoblasts or myotubes were incubated with 500 nm of library
compounds for 2 h before imaging. b) Chemical structures of probes
I25 and I31. c) Fluorescent images of I25 and I31 before (right) and
after (left) muscle differentiation. Scale bar=20 mm.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201007626.
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2761

Communications
strongest band at 54 kDa completely disappeared upon
competition with unmodified I25 or I31, but not with rhod-
amine 123 or rhodamine B, which were included as structur-
ally similar controls. Thus, we concluded that the 54 kDa band
corresponded to the most convincing binding target protein of
the compounds. The band was excised, sequenced, and
identified to be tubulin (see the Note 1 in the Supporting
Information).
While the affinity pull-down assay identified the major
binder to be tubulin, a well-known cytosolic protein, our
compounds appeared to be localized to mitochondria in live
cells. Affinity-based isolation greatly depends on protein
abundance as well as the protein-binding affinity. Since
tubulin is a highly abundant protein in cells (10–20 mm),[15]
its isolation might be an artifact. Therefore, we further
explored the endogenous binding protein in the context of
live cells.
For live-cell investigation, we synthesized a cell-perme-
able chemical-affinity probe, which has a thiol-reactive
chloroacetyl group, to enable covalent binding to target
proteins (Figure 2c). The compound is named CDy2 (Com-
pound of Designation yellow 2), following the biological
convention of Cluster of Designation (CD) for cell-surface
markers. The benefit of the chemical-affinity probe is that
once it forms a covalent bond with its targets in live cells,
those labeled proteins can be visualized by scanning the SDS-
PAGE gel with a fluorescence scanner, even though the
proteins are denatured.
When applied to myoblasts and myotubes, CDy2 showed
a 2.3-fold increase in fluorescence intensity after differentia-
tion [(2.3 Æ 0.4)-fold increase, N = 3; Figure 2d], which is
comparable to the increases observed with I25 and I31. To
unveil the endogenous binding protein(s), myoblasts or
myotubes were incubated with CDy2 for 1 hour. Labeled
lysates were separated by SDS-PAGE and analyzed with a
fluorescence scanner (Figure 2d). Again, a unique fluores-
cently labeled band was observed around 54 kDa (Figure 2e).
Also, pretreatment of myotubes with I31 reduced the
intensity of the CDy2-labeled protein band, indicating
effective competition of CDy2 with I31 in live cells (Fig-
ure S3b). To identify the labeled protein, cell lysates were
separated by two-dimensional gel electrophoresis, and fluo-
rescently labeled spots around 54 kDa were excised and
sequenced (Figure 2 f; see Note 2 in the Supporting Informa-
tion). To our surprise, the major spots were found to be
mitochondrial aldehyde dehydrogenase (ALDH2), not tubu-
lin.
Figure 2. Identification of protein binders in vitro and in living cells.
a) Chemical structure of the affinity matrix used to isolate cell proteins.
b) SDS-PAGE analysis of bead-bound proteins from C2C12 myotubes.
In a competition assay, myotube lysates were pre-incubated with
100 mm of I25, I31, or control compounds (rhodamine 123 and
rhodamine B) before the affinity pull-down experiment. c) Chemical
structure of CDy2 for labeling a target protein in living cells.
d) Myoblasts (MB) or myotubes (MT) were incubated with CDy2
(500 nm) for 30 min and imaged with a fluorescent microscope. Then,
cells were lysed for in-gel fluorescence analysis (lex =530 nm,
lex =580 nm) (e) Western blot analysis (WB). f) Two-dimensional gel
analysis for the identification of the labeled protein.
To validate the binding in live cells, firstly ALDH2 or
tubulin was overexpressed in HEK cells. Each protein was
tagged with green fluorescent protein (GFP) to distinguish it
from the endogenous proteins. Forty-eight hours after trans-
fection, cells were labeled with CDy2 and each lysate was
subjected to SDS-PAGE analysis (Figure 3a); the HEK293
cell line was chosen because of its relatively high transfection
efficiency. The result shows that CDy2-labeled ALDH2-GFP,
but not tubulin-GFP. Secondly, ALDH2 expression was
suppressed by small interfering RNA (siRNA; Figure 3b).
Upon ALDH2 knock-down, the CDy2-labeled band (54 kDa)
was dramatically reduced. These siRNA and overexpression
down assay is the conventional method for identifying small-
molecule-binding proteins.[14] Affinity resins were incubated
with myotube lysates and washed with buffer to remove
nonspecific binders. Then, resin-bound proteins were sepa-
rated by SDS-PAGE and stained with Coomassie blue
(Figure 2b). One enriched protein band at approximately
54 kDa was observed along with several other bands.
To determine the specificity of protein binders to these
compounds, we conducted a competition assay. Myotube cell
lysate was pre-incubated with 100 mm of I25, I31, rhod-
amine 123, or rhodamine B before affinity pull-down. The
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ical properties. Once a small molecule is sequestered in an
organelle such as mitochondria, its interactions with
proteins are greatly limited within the cellular compart-
ment. In this study, CDy2 showed an apparent binding
affinity to tubulin in vitro but binded to ALDH2 in live
cells. This implies that CDy2 molecules are sequestered in
mitochondria rapidly before they have a chance to react
with tubulin in the cytoplasm. Elucidating the protein that
binds the small molecule is the most challenging part of
chemical genetics. Our observations give an important
warning that the binding partner should be carefully
evaluated in the context of the environmental factors.
Figure 3. Validation of the identity of the labeled protein in living cells.
a) GFP-tagged ALDH2 or tubulin constructs were transfected into HEK293
cells. After 48 h, cells were labeled with CDy2 and subjected to in-gel
fluorescence imaging. Immunoblot analysis shows the endogenous (black
arrow) and over-expressed (red arrow) proteins. b) C2C12 myoblasts were
transfected with siRNA against ALDH2. After 72 h of transfection, cells
were labeled with CDy2. Fluorescence labeling patterns of CDy2 were
directly compared to ALDH2 immunofluororescence (green).
Received: December 6, 2010
Keywords: fluorescent probes · imaging agents ·
.
live-cell imaging · mitochondria · rosamine
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selectivity to rosamine probes deserves careful consideration.
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