Self-catalyzed immobilization of GST-fusion proteins for genome-encoded biochips

Article abstract of DOI:10.1021/bc400128g

With the surge of proteomic information that has become available in recent years from genome sequencing projects, selective and robust technologies for making protein biochips have become increasingly desirable. Herein, we describe the development of small-molecule SNAr electrophiles (smSNAREs), a new class of capture probes that enables a selective, single-step immobilization for protein biochips. This enzymology-driven approach rides on the binding and catalytic mechanism of SjGST. We have designed and synthesized mechanism-based substrate analogs 3, 4, and 5 as electrophilic precursors for conjugation of glutathione S-transferase (GST) or any of its fusion proteins. Upon evaluating the conjugation of these probes to glutathione in the presence of SjGST via UV-visible spectroscopy (UV-vis) and LC-MS techniques, we found that 3, 4, and 5 were transferable to GSH. Through the anchoring of alkyne 5 as a smSNARE probe on glass surface, we demonstrate the single-step, self-catalyzed immobilization of SjGST. Fluorescence imaging quantitatively revealed an 18-fold increase in selective binding of SjGST over random orientations (due to nonspecific binding) of the protein. Binding between GST and smSNARE surface is robust and does not reverse upon adding up to 100 mM GSH. Further, a 6-fold increase in resolution for the smSNARE surface probe was observed over commonly employed commercially available GSH-epoxy surfaces. Detailed control experiments revealed insights into the reversibility of binding and catalysis of GSH to form conjugation products with 5 in the presence of the enzyme. As an application of this protein capture technology, we printed alkaloid biosynthesis enzyme, isonitrile synthase (IsnA), to result in a biochip. Because proteins bearing a GST-fusion purification tag are commonly created through the pGEX expression system, these findings show broad potential applicability to genome-wide studies and proteomic platforms.

Full text of DOI:10.1021/bc400128g

Communication
pubs.acs.org/bc
Self-Catalyzed Immobilization of GST-Fusion Proteins for Genome-
Encoded Biochips
Alden E. Voelker† and Rajesh Viswanathan*,†
†Department of Chemistry, Case Western Reserve University, Millis Science Center: Rm 216, 2074 Adelbert Road, Cleveland Ohio
44106-7078, United States
S
* Supporting Information
ABSTRACT: With the surge of proteomic information that has become available in recent years from genome sequencing
projects, selective and robust technologies for making protein biochips have become increasingly desirable. Herein, we describe
the development of small-molecule SNAr electrophiles (smSNAREs), a new class of capture probes that enables a selective,
single-step immobilization for protein biochips. This enzymology-driven approach rides on the binding and catalytic mechanism
of SjGST. We have designed and synthesized mechanism-based substrate analogs 3, 4, and 5 as electrophilic precursors for
conjugation of glutathione S-transferase (GST) or any of its fusion proteins. Upon evaluating the conjugation of these probes to
glutathione in the presence of SjGST via UV−visible spectroscopy (UV−vis) and LC-MS techniques, we found that 3, 4, and 5
were transferable to GSH. Through the anchoring of alkyne 5 as a smSNARE probe on glass surface, we demonstrate the single-
step, self-catalyzed immobilization of SjGST. Fluorescence imaging quantitatively revealed an 18-fold increase in selective binding
of SjGST over random orientations (due to nonspecific binding) of the protein. Binding between GST and smSNARE surface is
robust and does not reverse upon adding up to 100 mM GSH. Further, a 6-fold increase in resolution for the smSNARE surface
probe was observed over commonly employed commercially available GSH-epoxy surfaces. Detailed control experiments
revealed insights into the reversibility of binding and catalysis of GSH to form conjugation products with 5 in the presence of the
enzyme. As an application of this protein capture technology, we printed alkaloid biosynthesis enzyme, isonitrile synthase (IsnA),
to result in a biochip. Because proteins bearing a GST-fusion purification tag are commonly created through the pGEX
expression system, these findings show broad potential applicability to genome-wide studies and proteomic platforms.
iniaturized biochips have revolutionized system-wide,
high-throughput studies of biological events.1−3 Rapid
the development of a new enzyme-catalyzed approach to
selectively conjugate proteins.
M
increase of available genomic information during the past two
decades has inspired the development of robust biochip
technologies for the elucidation of biochemical functions of
unknown proteins in a miniaturized format.4−6 Among these
methods, label-free conjugation techniques, especially of
commonly employed fusion proteins, add tremendous power
to the toolbox of chemical biologists.7 Label-free conjugation
through controlled fundamental mechanisms of protein-side
chain modifications is extremely powerful, yet challenging.8−13
Enzyme-catalyzed approaches offer unique advantages over
nonenzymatic chemical modifications due to the fact that
selectivity of conjugation is exquisitely programmed thereby
minimizing randomness and nonspecific modifications.14−16
Such techniques eventually result in superior readability of data
sets irrespective of nature of the application. We herein report
Through this strategy, glutathione S-transferase (GST) or its
fusion proteins are printed in a single step, utilizing the dual
properties of catalysis and binding of GST (Figure 1). The
SNAr (substitution-nucleophilic aromatic) reaction of Gluta-
thione S-Transferase (GST) offers a bioconjugation mechanism
to selectively print proteins as biochips with controlled
orientation. We describe herein the development of mecha-
nism-based small-molecule-SNAREs (SNAr electrophiles), a new
class of capture probes that lay the foundation for an enzymatic
strategy, for protein bioconjugation (and different from SNAP
Received: March 8, 2013
Revised: July 20, 2013
Published: July 24, 2013
© 2013 American Chemical Society
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Figure 1. Outline of smSNARE-captured bioconjugation strategy. The self-catalysis and binding to immobilize GST in their native orientation is
represented through participation of GSH as a nucleophile, smSNARE electrophile, and GST or its fusion protein as the enzyme.
a
Scheme 1
a
(A) GSH-conjugation to 1, the first step in mercapturic acid biosynthesis catalyzed by GST-A. (B) Design and electronic tuning of aromatic
substituents for the synthesis and evaluation of smSNARE probes 3, 4, and 5. Huckel charge densities are calculated using Chem 3D. (C) In vitro
̈
enzymatic incorporation of smSNARE probe 5 to GSH.
Receptors17). By combining the organic chemistry of this
smSNARE-capture strategy with molecular biology of pGEX
expression vectors, alkaloid biosynthesis enzyme IsnA is
selectively printed as a biochip in one step.
GSTs (E. C. 2.5.1.18) are a multigene family of enzymes that
are widely used as fusion tags to selectively isolate proteins
from proteomic mixtures.18 These enzymes are unique, because
in addition to binding to glutathione (GSH), a useful feature
for protein purification, they act as catalysts for the conjugation
of GSH to a wide variety of electrophiles.19−23 Nature has
evolved a suite of GSTs for detoxification chemistries that
comprise the mercapturic acid biosynthesis pathway in
eukaryotes. Binding studies by Mannervik (Scheme 1A) using
equilibrium dialysis methods on GST-A class enzymes with
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a
Scheme 2
a
(A) smSNARE surface probe capture of SjGST. (B) Plot consisting of signal intensities for fluorescence image of immobilized SjGST biochip
(ImageJ was used for quantification). Inset: biochip image of SjGST. + = GSH, GST are spotted followed by primary and secondary antibody.
Control = spots where all but primary antibody were added.
GSH and a 3,4-dichloro-1-nitrobenzene electrophile (1) reveal
that GST-A binds the GSH-conjugate 2 with >20-fold higher
affinity than GSH alone (Kd = 350 nM versus Kd = 7.1 μM,
respectively).20 Based on this observation, we hypothesized that
the development of a “first-generation” library of transferable
analogues (derived from the 1-chloro, 2,4-dinitrobenzene
(CDNB) scaffold) may lead to a new self-catalytic class of
GST-binding surface probes. Furthermore, because fusions of
Schistosoma japonicum GST (SjGST) contain catalytic domains
similar to GST-A class enzymes, we projected that our
mechanism-based probes may trigger a self-catalyzing capture
event for uniformly and selectively anchoring GST-fusion
proteins to create biochips.
We designed, synthesized, and evaluated in vitro a first-
generation class of smSNARE probes based on the canonical
CDNB scaffold (Scheme 1B). To permit tethering with
appropriate linkers, the C4 nitro group of CDNB was
exchanged for an ester functionality, based on modification of
the carboxylic acid group of 4-chloro-3-nitrobenzoic acid
(CNBA) or 3,4-dinitrobenzoic acid (DNBA). We aimed to
maintain the electrophilicity of C1 in order to facilitate the
nucleophilic aromatic substitution (SNAr) between GSH and
catalyzed conjugation reactions of these analogues to GSH
were evaluated. All three substrates displayed favorable
solubility for bioconjugation and the presence of an alkyne in
smSNARE 5 enabled facile surface monolayer construction
through click chemistry.
Upon subjecting alkyne 5 (0.5 mM) to conjugation with
GSH (5 mM) in the presence of 300 nM SjGST in 20 mM
phosphate buffer, pH 7.0 at 25 °C, we confirmed the formation
of GSH-5 (Scheme 1C). The UV−visible spectroscopic profile
of the conjugation reaction between GSH and compound 5
showed a new absorption peak appearing at ∼360 nm,
analogous to the absorption peak at 340 nm known to appear
when the canonical substrate CDNB is conjugated to GSH.24
The extinction coefficient at 356 nm (ε356) is 983 M−1·cm−1 for
conjugate GSH-5 (see Table S1). The presence of GSH-5 was
confirmed through detection of an LC-MS peak at m/z = 643,
consistent with the structure shown in Scheme 1C (Figure S2).
Similarly GSH conjugates of 3 and 4 were detected through
LC-MS analysis (Figure S5 and S8). Preliminary profiles of
reaction kinetics were obtained through a Lineweaver−Burk
plot analysis for conjugation reactions involving 3, 4, and 5,
respectively (Figures S3, S6, and S9). Transferability of these
analogues correlated well with reports on other substrate
analogs of CDNB.25,26 While the alcohol-bearing 3 displayed
superior transferability to GSH under SjGST catalysis, we chose
to study surface monolayer formation with alkyne 5 due to the
availability of an extended “clickable” tether. Azide-coated glass
surfaces were prepared by sequential treatment of ordinary
glass microscope slides with aminopropyl triethoxy silane
the electrophilic probes. Huckel charge densities at the chloro
̈
position for CDNB (+0.241) and CNBA (+0.231) were
comparable, indicating that the electrophilic nature at C1 was
preserved upon replacement of the C4 nitro group with a
carbonyl functionality. The synthesis and characterization of
three GST substrate analogues (compounds 3, 4, and 5)
followed straightforward methods (see SI). The SjGST-
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(APTES) and 6-azido-hexanoic acid. To confirm the presence
of azide groups on the slide, alkyne-bearing fluorescent
nanoparticles were conjugated to the azide-modified surface
under standard Cu(I)-catalyzed triazole formation conditions
(Figure S10). A greater signal enhancement was obtained for
nanoparticles clicked to smSNARE surfaces than using
fluorescent small-molecule click partners. Contact-angle mi-
croscopy (CAM) showed the increase in surface hydro-
phobicity through this functionalization (contact angle =
58.4°). Alkyne-containing smSNARE probe 5 was clicked to
the azido surface under similar conditions.
Conditions for the smSNARE-captured protein immobiliza-
tion were extended from the in vitro enzymatic reaction
previously performed in solution (Scheme 1C). The results of
this study are illustrated in Scheme 2. Enzymatic reaction
solution containing GSH (5 mM) and SjGST (300 nM) in
phosphate buffer (20 mM, pH 7.0) was applied to the
smSNARE probe surface (Scheme 2A). Similar to techniques
used in standard Western blots, the extent of the self-catalyzed
conjugation and binding between the smSNARE probe, GSH,
and SjGST was visualized by fluorescence immunolabeling with
a mouse anti-GST 1° and a fluorescently labeled 2° detection
antibody. Reproducibility in protein immobilization was evident
between each row as the spots are quadruplicate immobiliza-
tions, averaged to give intensity values that are plotted in
Scheme 2B. While the dilution of 1° antibody was varied by
factors of 10 from 1:10 to 1:10 000, the fluorescently labeled 2°
antibody was used at a constant dilution (1:200). Fluorescent
spots were visualized with a Typhoon gel imager (532 nm
excitation) and quantified with ImageJ software, showing a
consistent increase in intensity (Scheme 2B) with increasing
concentration of mouse anti-GST 1° antibody. The images
shown were quantified and plotted as displayed in Scheme 2B.
Controls without added SjGST (shown in Scheme 2B, right)
displayed no residual fluorescence signal, consistent with our
expectation that catalysis and binding requires the protein.
Scalability was not tested in this pilot study, though we envision
programming of this immobilization to be a smooth operation
at larger amounts of proteins.
In order to quantitate the level of GST immobilization
achieved through this smSNARE-capture step and to compare
it to the level of nonspecific adsorption of GST to glass surfaces
via different covalent and noncovalent binding methods, a
series of control experiments was carried out. Measured
intensities of immobilized SjGST were obtained through
methods identical to the previous biochip analyses (Scheme
2B). Lanes 1−4 (Figure 2) show immobilization where the
presence or absence of SjGST and GSH was tested individually.
The signal intensity in lane 2 confirmed that SjGST is required
for fluorescence. Lane 3 shows the SjGST nonspecific binding
to smSNARE probes without GSH conjugation, with the
intensity ratio of lane1:lane 3 showing the extent of selective
smSNARE-capture. An 18-fold increase in immobilization
efficiency is directly attributable to smSNARE-captured self-
catalysis. We attribute this 18-fold increase in SjGST binding to
the greater binding recognition (lowering of Kd) of SjGST to
the product of the conjugation reaction (corroborating with
reversibility study, see below). The lack of fluorescence
intensity in lane 4 confirmed that the antibodies were not
causing false positive fluorescence signals. Results from lanes
1−4 indicated that smSNARE-GSH conjugate, the product of
the GST-catalyzed reaction, has a much lower Kd than free
GSH. We wondered whether competitive binding might be an
Figure 2. Plot consisting of signal intensities for fluorescence image of
immobilized SjGST (ImageJ was used for quantification). Control
experiments comparing smSNARE probe surface to epoxy surfaces.
issue with the 5 mM concentration of GSH present during the
immobilization step. Also, as a corollary, we wondered if there
is a range of GSH concentration that might render reversibility
to the biochip formation step. In order to address this, we
carried out, post-immobilization, incubation of GST-labeled
smSNARE surfaces with either 100 mM GSH in phosphate
buffer (20 mM, pH 7.0) or 1% sodium dodecyl sulfate in
glycine buffer (200 mM, pH 2.0). In neither case did we
observe reversal of the binding of GST to the surface, indicated
by persistence of fluorescent signals at the same intensity before
and after these incubations.27 These observations point to the
fact that binding efficiency between GSH−smSNARE con-
jugate and SjGST increases drastically upon completion of a
single catalytic turnover, leading to a near-complete binding of
product. Surface plasmon resonance (SPR) studies in the future
may address quantitation of these observations. With respect to
reversibility, smSNARE technology offers a complementary
option to elegant mechanisms of reversible GST immobiliza-
tion reported by the Maynard group.28
Next, we addressed fundamental comparisons between
smSNARE-based immobilization and other commonly em-
ployed SjGST binding methods, such as GSH-modified epoxy
surfaces. For GST over epoxy-GSH surface, the immobilization
steps causing printing of proteins were performed consistently
with those recommended by the manufacturer (see SI). Also,
concentrations and duration for immobilization on epoxy
surface were adjusted to account for greater surface coverage of
epoxides than smSNARE slides. SjGST does indeed bind to
GSH-coated epoxy slides, as shown by the image in lane 5 of
Figure 2. Upon comparing lane 5 to lane 1, it was found that
smSNARE-capture strategy leads to a 6-fold improvement over
the standard GSH-epoxy surface for immobilization of GST.
Also, when no GSH is present (lane 7), SjGST binds to epoxy
slides in random orientations through nonselective modifica-
tion of its surface nucleophilic residues (e.g., lysine or cysteine),
indicated by low fluorescence intensity. The intensity ratio of
lane 1:lane 3 (178.22:9.81 = 18.1) versus that of lane 5:lane 7
(29.42:7.88 = 3.7) reflects the magnitude of signal-to-noise for
smSNARE versus epoxy surfaces, respectively. The smSNARE
probes therefore offer improved efficiency for selective
immobilization over regular epoxy surfaces. These observations
are consistent with previous studies, such as Mahal et al.’s
report that lectins oriented to succinimidyl-GSH surfaces
display a 17-fold increase in efficiency of protein immobiliza-
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Figure 3. AFM image showing morphology (left) and estimation of surface dimensions for immobilized SjGST (right).
tion.29 In this context, these controls correlate with findings
reported by Maynard et al. on GST immobilization through
chemically modified GSH analogues.28
identical conditions as described for SjGST. Scheme 3C shows
a fluorescence image of an immobilized GST-IsnA array that
has been treated with a mouse anti-GST 1° and a fluorescently
labeled 2° detection antibody. Control experiments were run
such that GST-tag bearing proteins are detected specifically by
the immunolabeling technique. Therefore, any of the proteins
from pGEX-3X vector (without the IsnA insert) containing a
fully expressed GST-tag will be detected. However, the SDS-
PAGE gel (SI) confirmed that the overexpressed IsnA-GST
fusion was the dominant GST-containing protein for “pull-
down” experiments after purification. The IsnA-GST fusion was
captured by both smSNARE surface and GSH-epoxy slides;
however, it was noted that the GSH-epoxy slides showed
greater background binding reflected by the intensity shown by
the fluorescent antibodies.27 This may imply that a significant
portion of the observed fluorescence of isnA-GSH-epoxy
system is attributable to nonspecific binding of the antibodies,
whereas IsnA-smSNARE surfaces suffer from this problem less,
and a more efficient IsnA capture occurs for the latter system.
The successful creation of this biochip opens the door for using
smSNARE-capture technology for building biocatalytic chips
with other genomic proteins and for lab-on-a-chip applications.
Future experiments will focus on deciphering enzyme action on
biochips of IsnA in addition to other genome-wide protein
profiling applications.
In conclusion, we report a new class of smSNAREs for a
single-step immobilization of GST-fusion proteins. Our initial
findings show that the transferable analogue 5 can serve to
irreversibly capture proteins with a 6-fold higher selectivity over
epoxy surfaces. The smSNARE probes used for this step offer a
mechanism-based protein immobilization tool toward proteo-
mic applications. Generally, in comparison to phenomenally
useful DNA biochips,31 protein biochip construction presents
unique challenges due to selectivity and preservation of enzyme
function. The SNArE-capture strategy is selective because it
takes advantage of the native catalysis of GSTs. Further the fact
that catalysis and binding occur in one step obviates the need to
prefunctionalize the surface with GSH. The latter feature is a
useful property for biochip applications, since GSH can
participate in aerobic chemistry (oxidation and elimination)
that causes degradation of the surface. In addition these
surfaces offer superior resolution over existing commercial
epoxy platforms. This strategy is envisioned as broadly
applicable to slides, surfaces, nanoparticles, and other
Atomic force microscopy (AFM) was employed to provide
insight into the surface morphology of GST-immobilized glass
surfaces prepared via the SNAr-E-capture method. Scheme 2E
shows an AFM image of a SjGST-immobilized slide prepared
with the same concentrations of GSH and GST as described
above. A 5 × 5 μm2 scan area was chosen in order to show
consistency of the immobilized biomolecules. This image shows
that the surface height varies between 0 and 5 nm depending
on the exact location on the slide. Using X-ray structure data
and a protein biophysical estimation suite (Phyre 2.0), the
diameter of surface-immobilized SjGST (see SI) was calculated
to be between 4.5 and 5 nm, which corresponds to the
morphology observed in the AFM image.
As an application of this smSNARE-capture technology, we
chose to immobilize an eDNA-derived (DNA recovered from
an environmental source) alkaloid biosynthesis protein of
interest (Scheme 3). The cDNA for isnA (responsible for
biosynthesis of L-tryptophan isonitrile30) was cloned into a
pGEX-3X vector and expressed under IPTG-induction to
produce GST-IsnA (see SI). The smSNARE-capture strategy
was employed for the immobilization of GST-IsnA under
a
Scheme 3
a
(A) Genome-encoded metabolic enzymes in alkaloid biosynthesis.
pGEX-3X-isnA was constructed for protein immobilization. (B)
Function of e-DNA-derived IsnA:biosynthesis of L-tryptophan
isonitrile. (C) SNARE-captured simultaneous self-catalysis and binding
to immobilize GST-IsnA.
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(9) Jonkheijm, P., Weinrich, D., Schroder, H., Niemeyer, C. M., and
Waldmann, H. (2008) Chemical strategies for generating protein
biochips. Angew. Chem., Int. Ed. Engl. 47, 9618−9647.
(10) Luk, Y. Y., Tingey, M. L., Dickson, K. A., Raines, R. T., and
Abbott, N. L. (2004) Comparison of the binding activity of randomly
oriented and uniformly oriented proteins immobilized by chemo-
selective coupling to a self-assembled monolayer. J. Am. Chem. Soc.
126, 9024−9032.
(11) Agarwal, P., van der Weijden, J., Sletten, E. M., Rabuka, D., and
Bertozzi, C. R. (2013) A Pictet-Spengler ligation for protein chemical
modification. Proc. Natl. Acad. Sci. U.S.A. 110, 46−51.
(12) Borra, R., Dong, D., Elnagar, A. Y., Woldemariam, G. A., and
Camarero, J. A. (2012) In-cell fluorescence activation and labeling of
proteins mediated by FRET-quenched split inteins. J. Am. Chem. Soc.
134, 6344−6353.
(13) Duppatla, V., Gjorgjevikj, M., Schmitz, W., Kottmair, M.,
Mueller, T. D., and Sebald, W. (2012) Enzymatic deglutathionylation
to generate interleukin-4 cysteine muteins with free thiol. Bioconjugate
Chem. 23, 1396−1405.
(14) Viswanathan, R., Labadie, G. R., and Poulter, C. D. (2013)
Regioselective covalent immobilization of catalytically active gluta-
thione S-transferase on glass slides. Bioconjugate Chem. 24, 571−577.
(15) Gauchet, C., Labadie, G. R., and Poulter, C. D. (2006) Regio-
and chemoselective covalent immobilization of proteins through
unnatural amino acids. J. Am. Chem. Soc. 128, 9274−9275.
(16) Yin, J., Liu, F., Li, X. H., and Walsh, C. T. (2004) Labeling
proteins with small molecules by site-specific posttranslational
modification. J. Am. Chem. Soc. 126, 7754−7755.
biomedical sensors that potentially can be layered with
smSNAREs. Proteomics applications also call for generality
for construction of ELISA-like “pull down” assays for a wide
range of proteins. The tool developed here anchors any gene of
interest to be bioconjugated in a single step after translation to
corresponding GST-fusion proteins. Therefore, future studies
will probe application of this strategy in ELISA-like binding
assays of proteomic mixtures.
ASSOCIATED CONTENT
* Supporting Information
■
S
Methods for the synthesis and spectral data of smSNARE
probes 3, 4, and 5. Data for enzymatic evaluation and detection
of GSH conjugates of 3, 4, and 5. Microscopy images of
surfaces, general protocols for immobilization, and additional
controls for GST immobilization. Cloning and biochemical
steps for preparation and expression of pGEX-isnA. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: rajesh.viswanathan@case.edu.
■
Notes
The authors declare no competing financial interest.
(17) Sollner, T., Whitehart, S. W., Brunner, M., Erdjumentbromage,
H., Geromanos, S., Tempst, P., and Rothman, J. E. (1993) SNAP
receptors implicated in vesicle targeting and fusion. Nature 362, 318−
324.
(18) Frangioni, J. V., and Neel, B. G. (1993) Solubilization and
purification of enzymatically active glutathione S-transferase (pGEX)
fusion proteins. Anal. Biochem. 210, 179−187.
(19) Mannervik, B., and Jensson, H. (1982) Binary combinations of
four protein subunits with different catalytic specificities explain the
relationship between six basic glutathione S-transferases in rat liver
cytosol. J. Biol. Chem. 257, 9909−9912.
(20) Jakobson, I., Warholm, M., and Mannervik, B. (1979) The
binding of substrates and a product of the enzymatic reaction to
glutathione S-transferase A. J. Biol. Chem. 254, 7085−7089.
(21) Moron, M. S., Depierre, J. W., and Mannervik, B. (1979) Levels
of glutathione, glutathione reductase and glutathione S-transferase
activities in rat lung and liver. Biochim. Biophys. Acta 582, 67−78.
(22) Armstrong, R. N. (1997) Structure, catalytic mechanism, and
evolution of the glutathione transferases. Chem. Res. Toxicol. 10, 2−18.
(23) Ji, X., Armstrong, R. N., and Gilliland, G. L. (1993) Snapshots
along the reaction coordinate of an SNAr reaction catalyzed by
glutathione transferase. Biochemistry 32, 12949−12954.
(24) Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974)
Glutathione S-transferases. The first enzymatic step in mercapturic
acid formation. J. Biol. Chem. 249, 7130−7139.
(25) Fujikawa, Y., Urano, Y., Komatsu, T., Hanaoka, K., Kojima, H.,
Terai, T., Inoue, H., and Nagano, T. (2008) Design and synthesis of
highly sensitive fluorogenic substrates for glutathione S-transferase
(GST) and application for activity imaging in living cells. J. Am. Chem.
Soc. 130, 14533−14543.
ACKNOWLEDGMENTS
■
The authors thank CWRU for funding this study. Authors
thank Drs. Dale Ray (CWRU, NMR) and Jon Karty (IU, MS)
for providing instrumentation support. Authors thank Prof. C.
Dale Poulter (Utah) and Dr. Jin Soo Seo (Utah) for generously
gifting chambers for slide modifications. Authors thank
̀ ́
Zhengao Mao and Prof. Genevieve Sauve (CWRU) for
discussions on AFM imaging. Authors thank Prof. Liming Dai
(CWRU) for contact angle measurements. Authors thank Prof.
Guillermo R. Labadie (IQUIR-CONICET) and Dr. Sucharita
Kundu (Utah) for discussions. Authors thank Prof. Gregory
Tochtrop (CWRU) for support with LC-MS analyses. Authors
thank Dr. Ulatowski (CWRU) and Prof. John Mieyal (CWRU)
for generous gift of anti-GST antibody.
REFERENCES
■
(1) Phizicky, E., Bastiaens, P. I. H., Zhu, H., Snyder, M., and Fields, S.
(2003) Protein analysis on a proteomic scale. Nature 422, 208−215.
(2) Zhu, H., and Snyder, M. (2003) Protein chip technology. Curr.
Opin. Chem. Biol. 7, 55−63.
(3) Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A.,
Bertone, P., Lan, N., Jansen, R., Bidlingmaier, S., Houfek, T., Mitchell,
T., Miller, P., Dean, R. A., Gerstein, M., and Snyder, M. (2001) Global
analysis of protein activities using proteome chips. Science 293, 2101−
2105.
(4) MacBeath, G. (2002) Protein microarrays and proteomics. Nat.
Genet. 32, 526−532.
(26) Lo, W.-J., Chiou, Y.-C., Hsu, Y.-T., Lam, W. S., Chang, M.-Y.,
Jao, S.-C., and Li, W.-S. (2007) Enzymatic and nonenzymatic synthesis
of glutathione conjugates: application to the understanding of a
parasite’s defense system and alternative to the discovery of potent
glutathione S-transferase inhibitors. Bioconjugate Chem. 18, 109−120.
(27) Voelker, A. E. Selective Fusion-Tag-Catalyzed Protein Immobiliza-
tions For Microarray and Biosensor Applications, Ph. D. Thesis, Case
Western Reserve University, Cleveland, OH, August 2013.
(28) Kolodziej, C. M., Chang, C.-W., and Maynard, H. D. (2011)
Glutathione S-transferase as a general and reversible tag for surface
immobilization of proteins. J. Mater. Chem. 21, 1457−1461.
(5) MacBeath, G., and Schreiber, S. L. (2000) Printing proteins as
microarrays for high-throughput function determination. Science 289,
1760−1763.
(6) Johnsson, N., and Johnsson, K. (2003) A fusion of disciplines:
chemical approaches to exploit fusion proteins for functional
genomics. ChemBioChem 4, 803−810.
(7) Yeo, W. S., Min, D. H., Hsieh, R. W., Greene, G. L., and Mrksich,
M. (2005) Label-free detection of protein-protein interactions on
biochips. Angew. Chem., Int. Ed. Engl. 44, 5480−5483.
(8) Stephanopoulos, N., and Francis, M. B. (2011) Choosing an
effective protein bioconjugation strategy. Nat. Chem. Biol. 7, 876−884.
1300
dx.doi.org/10.1021/bc400128g | Bioconjugate Chem. 2013, 24, 1295−1301

Bioconjugate Chemistry
Communication
(29) Propheter, D. C., Hsu, K.-L., and Mahal, L. K. (2010)
Fabrication of an oriented lectin microarray. ChemBioChem 11, 1203−
1207.
(30) Brady, S. F., and Clardy, J. (2005) Cloning and heterologous
expression of isocyanide biosynthetic genes from environmental DNA.
Angew. Chem., Int. Ed. Engl. 44, 7063−7065.
(31) Schena, M., Shalon, D., Davis, R. W., and Brown, P. O. (1995)
Quantitative monitoring of gene expression patterns with a
complementary DNA microarray. Science 270, 467−470.
1301
dx.doi.org/10.1021/bc400128g | Bioconjugate Chem. 2013, 24, 1295−1301