A Multipurpose Receptor Composed of ... - ACS Publications

Bioconjugate Chem. 2007, 18, 1935–1945

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A Multipurpose Receptor Composed of Promiscuous Proteins. Analyte Detection through Pattern Recognition Johan Viljanen,† Jenny Larsson,† Andréas Larsson,† and Kerstin S. Broo*,‡ IFM, Linköping University, S-581 83 Linköping, Sweden, and Department of Chemistry, Göteborg University, S-412 96 Göteborg, Sweden. Received July 5, 2007; Revised Manuscript Received August 17, 2007

A multipurpose receptor akin to the “electronic nose” was composed of coumarin-labeled mutants of human glutathione transferase A1. We have previously constructed a kit for site-specific modification of a lysine residue (A216K) using a thiol ester of glutathione (GSC-Coubio) as a modifying reagent. In the present investigation, we scrambled the hydrophobic binding site (H-site) of the protein scaffold through mutations at position M208 via random mutagenesis and isolated a representative library of 11 A216K/M208X mutants. All of the double mutants could be site-specifically labeled to form the K216Cou conjugates. The labeled proteins responded to the addition of different analytes with signature changes in their fluorescence spectra resulting in a matrix of 96 data points per analyte. Ligands as diverse as n-valeric acid, fumaric acid monoethyl ester, lithocholic acid, 1-chloro-2,4dinitrobenzene (CDNB), glutathione (GSH), S-methyl-GSH, S-hexyl-GSH, and GS-DNB all gave rise to signals that potentially can be interpreted through pattern recognition. The measured Kd values range from low micromolar to low millimolar. The cysteine residue C112 was used to anchor the coumarin-labeled protein to a PEG-based hydrogel chip in order to develop surface-based biosensing systems. We have thus initiated the development of a multipurpose, artificial receptor composed of an array of promiscuous proteins where detection of the analyte occurs through pattern recognition of fluorescence signals. In this system, many relatively poor binders each contribute to detailed readout in a truly egalitarian fashion.

INTRODUCTION The human olfactory system has inspired researchers to develop an electronic nose based on relatively nonspecific sensor electrodes (1, 2). The identification of a particular compound is then based on a mathematical interpretation of the signals from the array of electrodes (3, 4). Colorimetric sensor arrays for molecular recognition are also common in biosensing applications (5–8). We realized that this approach could be extended to utilize proteins as receptors and that the scaffolds we use in our protein reengineering efforts, the glutathione transferases (GSTs,1 E.C. 2.5.1.18) (41) are in fact exceptionally well suited for this type of experiment. Our previous studies have led us to focus on human glutathione transferase (hGST) A1-1 (Figure 1), a protein that is easy to express and purify, displays a high stability, and is tolerant to mutations (9–11). The crystal structure has been solved both with and without ligands (12, 13). The GSTs are dimeric detoxication enzymes (phase II) that catalyze the

Figure 1. Close-up of the crystal structure (1GUH) of wt hGST A1-1 (A216 replaced with K216) in a complex with S-benzyl-GSH. Positions 216 and 208 are shown in “stick” representation, and the flexible helix 9 (h9) is also indicated in the figure. The protein is dimeric but shown here as a monomer for reasons of clarity of presentation. The figure was done with the help of the PyMol (DeLano Scientific LLC) software.

* To whom correspondence should be addressed. Phone: +46-31772 2745. Fax: +46-31-772 3840. E-mail: [email protected]. † Linköping University. ‡ Göteborg University. 1 Abbreviations: A216KCou, coumarin-labeled A216K; C18, octadecyl; CDNB, 1-chloro-2,4-dinitrobenzene; FRET, fluorescence resonance energy transfer; G-site, glutathione-binding site; GS-thiol ester, thiol ester of glutathione; GSC, γ-Glu-Cys-Cys; GS-DNB, the conjugate formed between glutathione and CDNB; GSH, glutathione, γ-Glu-CysGly; GST, glutathione transferase; H-site, hydrophobic electrophile binding site; hGST A1-1, GST A1-1 isoform from human; HPLC, highperformance liquid chromatography; MALDI-MS, matrix assisted laser desorption ionization mass spectrometry; NA, immobilized NeutrAvidin; NaPi, sodium phosphate; NHS, N-hydroxysuccinimide; OD, optical density; PDEA, 2-(2-pyridinyldithio)ethaneamine; PEG, polyethyleneglycol; rt, room temperature; TFA, trifluoroacetic acid; TOF, time of flight; UV, ultraviolet; wt, wild type.

nucleophilic addition of the thiol of glutathione (GSH) to a wide range of hydrophobic electrophilic molecules (14). Since they have evolved to function as detoxication enzymes, GSTs have a promiscuous H-site that accepts a large variety of hydrophobic electrophiles (15–18), but the glutathione-binding site (G-site) is quite specific (13, 16). The modular features of the GSTs are ideal from a protein reengineering aspect with specific binding of GSH in the G-site combined with an adjacent, promiscuous H-site. In our previous studies we have extended the repertoire of the protein scaffolds by chemical modifications (19, 20). Some of the introduced moieties have fluorescent properties, and we recently developed a method for clean labeling through surfaceassisted delivery of fluorescent acyl groups to a specific lysine (K216) introduced through mutation of hGST A1 (21) (Scheme 1). The introduced K216 is located in the H-site of the folded

10.1021/bc700247x CCC: $37.00  2007 American Chemical Society Published on Web 10/17/2007

1936 Bioconjugate Chem., Vol. 18, No. 6, 2007

Viljanen et al.

Scheme 1

protein scaffold (Figure 1), and we hypothesized that K216Cou could report changes if the protein scaffold binds a molecule in its vicinity. In analogy to the electronic nose where the electrodes display broad and overlapping binding capacities to a number of substances (4), we needed to scramble the binding of different molecules by A216K. However, the overall fold of the protein and thus labeling of K216 by a thiol ester of glutathione (GSC-Coubio) need to be retained. We decided to make a focused library of K216/M208X mutants because position 208 is located in the H-site (Figure 1) and it has previously been shown to be of importance in the binding of the electrophilic substrate (22).

MATERIALS AND METHODS All chemicals and reagents used were of the highest purity available. The tripeptides were synthesized using solid-phase Fmoc chemistry, and protected amino acids were purchased from Novabiochem (Darmstadt, Germany) or Bachem Holding AG (Weil am Rhein, Germany). The fluorescent acyl groups were purchased from Sigma-Aldrich, Inc. (St. Louis, MO) or Molecular Probes, Inc. (Eugene, OR). EZ-Link PEO-iodoacetyl biotin and immobilized NeutrAvidin (NA) gel were purchased from Pierce Biotechnology, Inc. (Rockford, Il). All solid-phase syntheses were carried out in a VacMaster (International Sorbent Technology). The synthesis of GSC-Coubio has been described elsewhere (21). The HPLC experiments were carried out using a Kromasil C8 column (4.6 mm × 250 mm, Supelco, Inc.) or a Grace Vydac EVEREST C18 column (4.6 mm × 150 mm) attached to a Varian system with a ProStar 230 delivery system and a ProStar 330 photodiode array detector, controlled by the Varian LC Software. All mass spectra were recorded using a MALDI-TOF MS (Voyager System 4212, Applied Biosystems) with detection in the positive mode. The UV measurements were performed using a Varian Cay 100 Scan UV–visible spectrophotometer and the results analyzed with the CaryWin UV software. The fluorescence measurements were carried out on a Safire2 (Tecan), a SpectraMax M2 (Molecular Devices), or a Hitachi F-4500 fluorescence spectrophotometer. The degree of immobilization of labeled A216K on the hydrogel chip was

examined with an epifluorescence microscope (Zeiss Axiovert inverted microscope A200 Mot) equipped with a charge coupled device camera (Axiocam HRc, three chips). Site-Directed Mutagenesis, Protein Expression, and Purification. The vector pET 21 a (+) containing the hGST A1-1 mutant A216K gene was used as a template for replacement of M208 by site-directed mutagenesis using the Quik-Change site directed mutagenesis kit (Stratagene). Oligonucleotide, M208NNN forward (5′-GCCCAAGGAAGCCTCCCNNNGATGAGAAATCTTTAGAAG-3′), and its complement (M208NNN reverse) were designed in order to replace codon ATG (M208) with random nucleotide bases to create codons coding for different amino acids. The mutations were checked by DNA sequencing (GATC Biotech, Germany). From glycerol stocks of the different mutants, an amount of 200 µL of each was plated out on agar plates containing 100 µg/mL ampicillin and incubated at 37 °C over night. The next day the bacteria on the plates were resuspended in a few milliliters of 2YT-medium (16 g/L peptone, 10 g/L yeast extract, 5 g/L NaCl) and approximately half of the bacteria were pored into 1 L of 2YT medium containing 100 µg/mL ampicillin and incubated, shaking at 37 °C until the OD600 was about 0.6–0.7. The bacteria were induced to start producing the recombinant protein by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and incubating the mixture at 37 °C with shaking for 3.5 h. The cells were harvested by centrifugation (3000g, 30 min, 4 °C), and the pellets were resuspended in a protease inhibitor cocktail (Complete Mini EDTA-free, Roche) containing 20 mM NaPI, 0.02 % NaN3, pH 7, with one tablet per 10 mL of buffer. For lysing of the cells, freezing at -80 °C and thawing at 37 °C were performed four times. After the final thawing the lysates were centrifuged at 11000g for 30 min at 4 °C. The supernatants from the different cell lysates were desalted using PD-10 desalting columns (Amersham Biosciences). Purification of the different mutants was performed on a 3 × 5 mL HiTrap SP HP cation exchange column (Amersham ¨ KTA purifier system (Amersham Biosciences) attached to an A Biosciences). The column was equilibrated with 4 column

Fluorescent hGST A1 Mutants as Biosensors

volumes of buffer A (20 mM NaPi, 0.02% NaN3, pH 7). The eluates from the PD-10 columns were loaded onto the cation exchange column with a flow of 3 mL/min, and thereafter, the column was washed with another 3 column volumes of buffer A. The different recombinant hGST A1-1 mutants were eluted from the column in a linear 13 column volumes gradient at a flow rate of 2 mL/min expanding from 100% buffer A to 100% buffer B (buffer A with 1 M NaCl). From the gradient, 2 mL fractions of the eluted proteins were collected. The peak fractions were pooled and concentrated using Amicon Ultra-15 centrifugal filter units, 10000 MWCO (Millipore). Purity was confirmed by SDS–PAGE, and protein concentrations were determined spectrophotometrically using ε280 ) 24 700 M-1 · cm-1 (23). The activities of the GSTs were measured with a standard assay with 1 mM each of 1-chloro-2,4-dinitrobenzene (CDNB) and GSH (pH 6.5 in 0.1 M NaPi at 30 °C). The proteins were stored in NaPi buffered solutions containing NaCl and 10% glycerol at -80 or -20 °C until use in the experiments. Fluorescence measurements were performed on a Hitachi F-4500 fluorescence spectrophotometer, with excitation at 280 nm and emission scanning between 310 and 380 nm. Prior to the measurements, the proteins were diluted to 1 µM in 100 mM NaPi, pH 7.0, and the fluorescence spectra of A216K and wt hGST A1-1 were also collected for comparison purposes (Supporting Information). Preparative Labeling of A216K/M208X Mutants. The labeling reactions were performed in 200 µL mixtures of 20 µM protein, 0.1 M NaPi, pH 7, with 300 µM GSC-Coubio. The reaction was allowed to proceed at 25 °C for 5 h. For protease treatment, 2 µL of modification reaction mixture was diluted with 17 µL of 75 mM NH4OAc, pH 4, and the cleavage was started by adding1 µL of Staphylococcus aureus protease V-8 (1 µg/µL stock solution in 75 mM NH4OAc, pH 4). The mixture was then incubated at 25 °C and stopped by adding an equal amount of 0.2% TFA (e.g., 20 µL reaction mixture) after 2 h. Following incubation, 0.5 µL of the reaction mixture was mixed with 0.5 µL of saturated R-cyano-4-hydroxycinnamic acid matrix, and 0.5 µL of citrate (10 mg/mL) was also added to quench salts from the buffers. The samples were then analyzed by MALDI-MS with detection in the positive mode. Proteolytic digestion with this protease results in K216-containing fragments K216RKIFRF and EK216RKIFRF, corresponding to one missed cleavage. After modification was complete, 1.1 equiv of NA beads (binding capacity of g80 nmol of biotin/mL of gel) was added to the reaction mixture. The gel had been pre-equilibrated in 0.1 M NaPi, pH 7.1, containing 75 mM NaCl in VacMaster (International Sorbent Technology) tubes. After incubation with the gel (45 min), the labeled protein was eluted by a brief spin in a tabletop centrifuge (Scheme 1). Array Experiments with Addition of n-Valeric Acid, Fumaric Acid Monoethyl Ester, and Lithocholic Acid. A typical array experiment was performed in the following way: 300 µL mixtures of 1 µM labeled protein and 200 µM analyte (0.1 M NaPi, pH 7) were transferred to a 96-well black microplate (Nunc). A sample of each protein without analyte was also included. Each well was analyzed with respect to direct excitation of the coumarin residue at 355 nm and of the tryptophan residue at 280 nm. The emission was monitored from 370 to 500 nm. The fluorescence intensity at 408 nm was noted and plotted as a function of total analyte concentration. The equivalent parameters were also collected following addition of 200 µM GSH (20 mM stock solution) to each well containing the analytes. Affinity Determination Using Fluorescence Spectroscopy. Samples of coumarin-labeled mutants (1 µM in 0.1 M NaPi, pH 7.0) were set up in a 96-well half-area black microplate

Bioconjugate Chem., Vol. 18, No. 6, 2007 1937

with a clear bottom (Corning, Inc.). The analytes (GSH, CDNB, GS-DNB, or S-hexyl-GSH) were then added to the wells: GSH, CDNB, and GS-DNB in the range 10 µM to 1.5 mM and S-hexyl-GSH in the range 1 nM to 1.5 mM (Vtot ) 75 µL). The optical density (340–500 nm) was recorded for each well. In cases where the optical density exceeded 0.05 at the wavelength of excitation and emission, the fluorescence intensities were corrected according to the following equation (24):

(

Fcorr ) Fobs antilog

ODex + ODem 2

)

(1)

where Fobs and Fcorr are experimental values and corrected fluorescence intensities, respectively, and ODex and ODem are the optical densities of the sample at the wavelengths of excitation and emission, respectively. The dissociation constant Kd was determined by fitting the following equation to the experimental results under the assumption of a 1:1 binding model: Fobs )

Fbound[A] + FfreeKd [A] + Kd

(2)

where Fobs is the observed fluorescence intensity, Fbound is the fluorescence of the protein bound to the analyte, Ffree is the fluorescence of free protein, and [A] is the concentration of free analyte. The numerical value of [A] can be derived from the following: [A] )

[P]tot + Kd - [A]tot

2

+

√(

)

[P]tot + Kd - [A]tot 2

2

+ Kd[A]tot (3)

where [P]tot is the total concentration of protein and [A]tot is the total concentration of analyte. The fitting of the equations to the experimental values was done with the IGOR Pro 5.0 software (WaveMetrics, Inc.). Reproducibility of GSH Addition. The reproducibility was checked by splitting a sample of A216K/M208T (1 µM in 0.1 M NaPi, pH 7.0) in three different wells. As a control, GSH (200 µM) was added and the resulting coumarin fluorescence was recorded. The values differed by less than 5%. Immobilization of A216Kcou on a PEG-Based Biosensor Chip and Fluorescence Microscopy. A thiol self-assembled monolayer (SAM) was formed by incubating gold-coated silica substrates with 100 µM HS(CH2)11CONH(C2H4O)11CH3 (Polypure AS, Norway) in 99.5% ethanol for at least 24 h at room temperature. The thiol SAM was used as a platform for UV grafted polymerization of a polyethylene glycol (PEG) based matrix, using a process described in detail elsewhere (25, 26). By use of masks, arrays of matrix spots were prepared by irradiating the samples with a Philips TUV PL-L 18 W Hg lamp in the presence of a PEG methacrylate monomer (∼10 EG units) (PEG10MA) and 2-hydroxyethyl methacrylate (HEMA). Two different arrays of (i) 200 µm and (ii) 300 µm spots, both with a spacing of 50 µm, were irradiated for 6 and 4 min, respectively. The matrix spots were then carboxylated using bromoacetic acid (27). Both samples were activated for 20 min with an aqueous mixture of N-ethyl-N′-[3-(dimethylamino)propyl]carbodiimide (Biacore AB, Uppsala, Sweden) and Nhydroxysuccinimide (Sigma-Aldrich Sweden AB) at 0.2 and 0.05 M, respectively. A216Kcou was then immobilized on the hydrogel chip (i) via nucleophilic amino acid residues on the protein and (ii) via Cys112 subsequent to 30 min of incubation in 80 mM 2-(2-pyridinyldithio)ethaneamine in Milli-Q water. The concentration of A216Kcou was 0.5–1.0 µM in 10 mM sodium acetate buffer, pH 4.5, and the immobilization time was 60 and 40 min for steps i and ii,, respectively. In addition, sample i was deactivated with 1 M ethanolamine in Milli-Q


Viljanen et al. Scheme 2

Figure 2. MALDI-MS spectra of proteolytic (S. aureus V-8) peptide fragments from (A) A216K/M208T (10 µM) incubated with GSC-Coubio (100 µM) for 5 h and (B) A216K/M208E (10 µM) incubated with GSCCoubio (100 µM) for 5 h. The reaction has gone to completion, but the parent fragments would have an m/z of 994 g/mol as indicated by a dotted arrow. Modified fragments are highlighted with arrows. The peak at 992 g/mol corresponds to the sodium adduct of GSC-Coubio.

water for 30 min. The arrays were then examined with an epifluorescence microscope (Zeiss Axiovert inverted microscope A200 Mot) equipped with a charge coupled device camera (Axiocam HRc, three chips). A 10× magnification objective was used along with a 365/15 nm band-pass excitation filter and a 397 nm low-pass emission filter. The exposure time was 1.25 s, and the obtained images were enhanced with respect to color and contrast.

RESULTS Preparation of the A216K/M208X Library and Pilot Labeling of the Purified A216K/M208X Mutants. Eleven double mutants (A216K/M208L, F, S, Y, A, E, R, T, G, K, and C) were isolated and purified as described elsewhere (28). The purities were confirmed by SDS–PAGE, their specific activities toward the standard substrates CDNB and GSH were measured, and their fluorescence spectra resulting from excitation at W21 were recorded and compared to those of wt hGST A1-1 and A216K, respectively (Supporting Information). Pilot labeling experiments were performed in 20 µL aliquots with GSC-Coubio (100 µM) and purified protein (10 µM) in buffer (0.1 M NaPi, pH 7.0). The reactions were analyzed by MALDI-MS following proteolytic digestion. To simplify subsequent analyses, we used S. aureus V-8 protease because it cleaves after acidic residues and is thus unaffected by lysine modification. For control purposes, the proteins were also digested after incubation in buffer without addition of any reagent. The single mutant A216K was included in all experiments leading to a total of 12 proteins in the receptor family. All mutants became acylated by GSC-Coubio at residue K216 (Figure 2). Preparative Labeling of the Receptor Family. The 12 proteins were labeled on a preparative scale in solutions containing 20 µM protein and 300 µM GSC-Coubio in 0.1 M NaPi, pH 7.0, buffer. On the basis of the results from the pilot experiments, the reactions were allowed to proceed at 25 °C for 5 h. In all subsequent experiments, the labelling reactions were monitored by subjecting 2 µL of the reaction mixtures to proteolytic digestion with subsequent analysis by MALDI-MS. In order to remove used and excess reagent, an amount of 1.1 equiv of NA beads (binding capacity of g80 nmol of biotin/ mL of gel) was added to each reaction mixture after 5 h as described previously (21) (Scheme 1). After incubation with the gel for 45 min, the labeled protein was eluted by a brief

spin in a tabletop centrifuge. We have previously demonstrated that this treatment quantitatively removes residual reagent. After treatment with the NA beads, the fluorescence spectra of the protein eluates displayed the typical coumarin emission (Supporting Information). Addition of n-Valeric Acid, Fumaric Acid Monoethyl Ester, and Lithocholic Acid to the Receptor Family. In order to explore the signaling potential of the receptor family and find out what kind of information that could be extracted, a screening experiment was set up with 3 analytes (n-valeric acid, fumaric acid monoethyl ester, and lithocholic acid) (Scheme 2) and all 12 proteins (the double mutants and A216K) in a microplate format. The chosen analytes do not display any significant absorbance at the measured wavelengths and concentrations (data not shown). The fluorescence spectra of 12 proteins by 3 analytes plus those of the proteins without analytes, the analytes without proteins, and the buffer alone were thus collected (Figure 3). Each well was analyzed with respect to direct excitation of the coumaryl residue at 355 nm and also through excitation at 280 nm (W21) with concomitant FRET to the coumaryl residue. Excitation of GSC-Coubio at 280 nm did not result in any emission from the coumaryl moiety. In these experiments, both the changes in fluorescence intensities (∆FCou) and the positions of the emission maxima (∆λem,max) were documented. The equivalent parameters were also collected following addition of 200 µM GSH to each sample, resulting in a matrix of eight data points for each analyte and protein combination or 96 data points per analyte (Table 1). Affinity Studies. In order to investigate the capacity of binding, the Kd values were determined for a subset of analytes. In this experiment, labeled protein was titrated with analytes (all in 0.1 M NaPi buffer, pH 7.0) (Figures 5 and 6). The fluorescence spectra were recorded using λexc ) 295 nm (W21) and λexc ) 355 nm (K216Cou). In the case of GSH and S-hexylGSH, the spectra could be analyzed directly. However, both CDNB and GS-DNB absorb light at the wavelengths used for both excitation and emission. Before the results could be



Figure 3. Example of the screening experiment showing changes in fluorescence after addition of first lithocholic acid (200 µM) to wells containing 1 µM labeled A216KCou/M208R followed by GSH (200 µM). For each protein and analyte combination, excitation at 280 nm (W21) with concomitant FRET to the coumaryl residue was followed by direct excitation of the coumaryl residue at 355 nm. Table 1. Addition of n-Valeric Acid, Fumaric Acid Monoethyl Ester, and Lithocholic Acid to the Protein Array L

F

S

Y

A

∆λem,max,Cou ∆λem,max,FRET ∆λem,max,Cou,GSH ∆λem,max,FRET,GSH ∆FCou ∆FCou,FRET ∆FCou,GSH ∆FCou,FRET,GSH

0 1 0 0 8 –7 39 26

–1 –1 0 0 9 2 44 39

0 0 0 0 –1 –2 40 35

0 0 0 0 –1 –4 2 6


0 0 0 0 10 –3 39 28

0 0 0 0 14 9 47 43

0 0 1 0 –2 8 40 38

Fumaric –1 0 0 0 –3 0 4 8


n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m.


0 0 0 0 9 38 4 39

a

0 0 0 –10 –26 –2 –25 17

E

R

T

G

K

A216K

C

0 0 0 0 –3 –2 27 33

1 1 0 0 –1 0 35 34

–1 0 1 2 21 –2 31 25

0 –1 0 0 –8 –11 9 8

0 0 0 0 3 –2 66 39

0 0 n.m.a n.m. –1 –1 n.m. n.m.

Acid Monoethyl Ester 0 0 0 0 0 0 0 –1 0 0 0 0 –1 1 –1 3 4 5 36 22 28 39 19 36

0 0 0 0 –1 1 30 35

0 0 0 0 26 4 33 30

0 0 1 1 –2 –5 15 19

1 0 0 0 4 3 54 39

0 0 n.m. n.m. 1 4 n.m. n.m.

0 1 –2 0 25 44 21 53

0 0 0 –1 6 23 –3 34

0 0 0 0 1 20 –9 27

–1 0 –1 0 30 55 43 60

0 0 n.m. n.m. 5 48 n.m n.m

n-Valeric Acid 0 0 0 0 0 0 0 0 0 2 –1 1 37 22 38 17

Lithocholic Acid –2 1 0 –3 0 0 –3 0 7 0 48 47 33 29 52 54

1 –1 –3 –7 –34 47 6 53

n.m. ) not measured.

interpreted, the emission intensities were corrected using the approximate correction equation (24): Fcorr )

Fobs antilog(ODexc + ODem) 2

coupled device, and the obtained images were enhanced with respect to color and contrast (Figure 9).

DISCUSSION (4)

We found that the proteins bound GSH, S-hexyl-GSH, CDNB, and GS-DNB with Kd values spanning more than 3 orders of magnitude (from 2.5 µM to 6.9 mM) (Table 3). Immobilization of A216KCou on a Hydrogel Chip. The cysteine residue C112 (Figure 8) was utilized to couple A216KCou site specifically to a microarray hydrogel surface (Figure 9). Labeled protein (0.5–1.0 µM) was incubated together with the PDEA activated surface in 10 mM NaOAc buffer, pH 4.5, for 40 min. A216KCou was also immobilized on a NHSactivated surface via nucleophilic amino acids on the protein. The attachment of protein was verified by MALDI-MS, following direct proteolytic digestion (S. aureus V-8) on the array surface (Supporting Information). The arrays were then examined with an epifluorescence microscope equipped with a charge

This investigation was performed because we wanted to emulate the ideas of the “electronic nose” (1, 2, 4, 29, 30) using proteins instead of electrodes, thus getting access to the complex and vast amount of interactions between proteins and small molecules. The idea of having an array of mediocre binders that combine to form a module that can report the presence of a given molecule with a much higher degree of specificity is highly appealing from a scientific point of view, and it can conceivably be of some commercial interest in the future. The inherently relaxed substrate requirements, a hallmark of GST catalysis and a prerequisite for detoxication, together with our newly developed kit for user-friendly protein labeling (21) are the perfect combination for building a multipurpose, proteinbased sensor of this type. The task was to deform the binding site (the H-site) of A216K to an appropriate degree, not too much and not too little. Too


Figure 4. Graphic representation of the changes in fluorescence described in Table 2. Each protein to analyte combination gives rise to eight parameters. In order to simplify the interpretation, the diagrams show the differences in changes between (A) fumaric acid monoethyl ester and n-valeric acid and (B) lithocholic acid and n-valeric acid. Valeric acid has thus been used as a sort of standard or normalizing substance. The mutants A216K/M208F and L were omitted in the investigation with lithocholic acid.

much would perhaps result in nonexistent binding in the H-site and thus no analyte recognition or spoilage of the G-site with no labeling as a potential outcome. Too little would result in a protein array without binding diversity, somewhat like having an array with just one protein attached to the surface. The mutations also have to be relatively close (in the folded state) to position 216 so that K216Cou can sense and report changes upon binding. In addition, the new mutations also need to be located in or near the H-site where the ligands/analytes are supposed to bind. Previous studies concerning hGST A1-1 have reported that M208 can be mutated with altered substrate preferences as a result (22, 31). The side chain of M208 points into the H-site and is located in a region that presumably can undergo changes upon binding of electrophiles because helix9 changes its conformation upon binding of substrates (Figure 1). We wanted a varied panel with mutations ranging from negatively to positively charged, from small to large, from hydrophilic to hydrophobic, and with and without functional groups such as –OH or –SH. We therefore created a A216K/ M208X library using a randomized primer and sequenced, expressed, and purified enough clones to obtain 11 double mutants with a wide spread of properties. One of the clones,

Viljanen et al.

Figure 5. Addition of increasing amounts of GSH to (A) 1 µM A216KCou/M208F, (B) 1 µM A216KCou/M208E, and (C) 1 µM A216KCou/M208T. An equation that describes the dissociation of a bimolecular complex was fitted to the experimental data and gave Kd values of 72, 73, and 96 µM, respectively. Solid lines represent the best fits to the experimental results. The x-axis corresponds to the total amount of added GSH ([GSH]tot). At the concentrations shown here, [GSH]tot ≈ [GSH]free.

A216K/M208K, has an additional lysine residue in the H-site, and we therefore studied if that could also become acylated by GSC-Coubio or if the protein would only become singly labeled. The double mutants A216K/M208X can react with GSCCoubio to form K216Cou conjugates. We have previously shown that A216K can be treated with GSC-Coubio to form an A216KCou conjugate and that any used up or excess reagent can be quantitatively removed using NA beads relying on the biotin–streptavidin interaction (21) (Supporting Information). Fortunately, the double mutants retained this crucial capacity, enabling us to quick and easy labeling of the receptor family. The speed of the reaction varied, but incubation over 5 h resulted in >85% labeling for all but two mutants (Supporting Information), which we deemed high enough for our needs. We thus have a system where we can start with pure proteins (SDS–PAGE gels, Supporting Information) that can be labeled to more than 85% (Figure 2 and Supporting Information) and where there is no residual fluorescent labeling reagent left in solution after treatment with NA beads (HPLC chromatograms in Supporting Information). Similar analytes (n-valeric acid and fumaric acid monoethyl ester) result in different patterns. In order for this system to work, the receptor must be able to report “signature changes” for different molecules even if they appear similar from the


Figure 6. Addition of increasing amounts of (A) CDNB and (B) GSDNB to the parent protein A216Kcou (1 µM). The resulting Kd values were determined by fitting an equation that describes the dissociation of a bimolecular complex, to 1.3 mM and 347 µM, respectively. Solid lines represent the best fits to the experimental results. At the concentrations shown here, [analyte]tot ≈ [analyte]free.

viewpoint of a promiscuous GST. It is also an advantage if a totally dissimilar molecule (with respect to size, hydrophobicity, charge, etc.) gives a completely dissimilar response. In order to test this and also to obtain information about what parameters are exploitable and how substantial the changes in fluorescence would be, we set up a pilot experiment using all 12 proteins (A216K and the 11 A216K/M208X mutants) and 3 different analytes: n-valeric acid, fumaric acid monoethyl ester, and lithocholic acid (Scheme 2). These molecules are all soluble in buffer, and they do not have complicating absorbance spectra at the concentrations used in the experiments. The first two are quite small and have fairly similar structures, and the last one is a large, bulky steroid that is a naturally occurring bile acid known to be a liver-toxic metabolite (32, 33) and to probably bind in the groove between the subunits (34, 35). We also wanted to investigate whether W21 that is used to study folding of hGST A1-1 (11, 36) could report binding and whether the coumarin that is located in or near the H-site could report binding through direct excitation. The results from the FRET experiments showed competing mechanisms between energy transfer from W21 to the coumarin fluorophore and coumarin quenching. Since the values differ between ∆FCou and ∆FCou,FRET following blank reduction and upon analyte addition (Table 1) and also since the emission intensity influence for the coumarin is much more significant than the emission intensity influence for W21, we concluded that the parameter ∆FCou,FRET includes both information contributed from coumarin quenching and distance changes between W21 and coumarin. Addition of GSH to GSTs can affect the binding of a second substrate in the H-site, and we therefore added GSH to each well after collecting the data in the analyte plus protein experiment. We are of course aware of the fact that GSH alone can cause changes in the fluorescence of the coumarin moiety (21) and that the overall signal we observe upon addition of GSH is a combination of both effects. On a cautionary note, GSH can also form adducts with some electrophilic substances, so it should be used with discretion. One final parameter that


we were interested in was the change in position of λem,max. In the literature, coumarin derivatives are reported to change their emission maxima up to 14 nm going from CHCl3 to MeOH as a solvent (24). In all, this set of parameters (∆λem,max,Cou, ∆λem,max,FRET, ∆λem,max,GSH, ∆λem,max,FRET,GSH, ∆FCou, ∆FCou,FRET, ∆FCou,GSH, ∆FCou,FRET,GSH) resulted in a matrix of 12 proteins by 8 data points, or a total of 96 measured parameters for each analyte (Table 1, Figure 4). In an effort to aid visualization, the data from fumaric acid monoethyl ester and lithocholic acid were compared to the changes obtained when n-valeric acid was added. This choice was simply based on the fact that n-valeric acid is the analyte with the least amount of functional groups in the test set. We were pleased to observe that the biosensor panel could differentiate between n-valeric acid and fumaric acid monoethyl ester (Table 1). The numbers were relatively small, but that is perhaps to be expected when the analytes differ so little. These changes reported by a single protein or just a few proteins in an array would not amount to anything useful, but the combined effect from all 12 proteins is quite promising. On the other hand, adding lithocholic acid to the panel of proteins resulted in large changes in fluorescence. This molecule definitely affects both W21 and K216Cou in a completely different way than either of the two smaller ligands. Interestingly, we observed wavelength shifts of 10 nm for A216K/ M208Y, suggesting a quite substantial change in the hydrophobicity of the microenvironment of the coumarin moiety upon binding of lithocholic acid. It appears that the most common result when a ligand is added to the panel of labeled proteins is quenching of the fluorescence of coumarin. The most likely cause for this is static quenching; i.e., upon addition of the analyte a nonfluorescent complex is formed between the coumarin and the quencher that serves to limit the absorption by reducing the population of active, excitable molecules. There is a small variation in the degree of quenching of the coumaryl residue between the A216K/M208X mutants, probably resulting from differing microenvironments in the binding site leading to different orientations of K216Cou and analyte. Interestingly, when S-hexyl-GSH is added to A216Kcou, the fluorescence intensity actually increases (Figure 7) but no wavelength shift is observed. The wavelength shifts upon analyte addition are probably due to solvent or polarity effects. Control experiments demonstrated that GSC-Coubio in buffer has a significantly lower emission intensity and a λem, max that is red-shifted with 17 nm compared to A216Kcou (data not shown). This strengthens the notion that the coumaryl group is positioned in a hydrophobic environment in a relatively static manner when attached to K216, since no shift or small wavelength shifts are observed upon analyte addition (Table 1). The small shifts that we do observe in some cases are probably caused by a reorganization of the C-terminal helix upon analyte addition with concomitant alteration of the position of K216Cou from a hydrophobic environment (H-site) to a hydrophilic one (water outside H-site) or vise versa. No Apparent Correlation between Mutations and Signal Changes. The mutations in position 208 represent a wide range of functionalities. Despite this, there are no striking and consistent trends. In fact, the most striking result seems to be that there are no predictable trends regarding changes in fluorescence. The mutants do not seem to follow any rules, for example adding lithocholic acid to A216K/M208S, T, and Y results in ∆FCou values of 9, 25, and –26, respectively (Table 1). Another noteworthy example of this is that the ∆FCou changes recorded for E, R, and K208 are 0, -34, and 1,


Viljanen et al.

Table 2. Differences between Fumaric Acid Monoethyl Ester and n-Valeric Acid (∆ F–V) and between Lithocholic Acid and n-Valeric Acid (∆ L–V)a L

F

S

Y

A

0 0 1 0 –1 10 0 3

–1 0 0 0 –2 4 2 2

0 0 0 0 –1 4 –1 1

0 0 0 0 10 40 –36 4

0 0 0 –10 –25 2 –27 11

–2 0 0 –3 7 49 –4 14

E

R

T

G

K

A216K

C

0 0 0 0 2 7 1 3

–1 –1 0 0 0 1 –5 1

1 0 –1 –2 5 6 2 5

0 1 1 1 6 6 6 11

1 0 0 0 1 5 –12 0

0 0 n.m.a n.m. 2 5 n.m. n.m.

1 –1 –3 –7 –31 49 –21 20

–1 0 –2 0 26 44 –14 19

1 0 –1 –3 –15 25 –34 9

0 1 0 0 9 31 –18 19

–1 0 –1 0 27 57 –23 21

0 0 n.m. n.m. 6 49 n.m. n.m.

∆ F–V ∆λem,max,Cou ∆λem,max,FRET ∆λem,max,Cou,GSH ∆λem,max,FRET,GSH ∆FCou ∆FCou,FRET ∆FCou,GSH ∆FCou,FRET,GSH

0 –1 0 0 2 4 0 2

1 1 0 0 5 7 3 4




0 0 –1 0 –1 3 0 2 ∆ L–V

a

1 –3 0 0 –2 46 7 37

n-Valeric acid acts as a standard comparison. n.m. ) not measured.

Table 3. Dissociation Constants of Complexes Formed from Labeled A216K Mutants and a Subset of Analytes, Determined by Fluorescence Spectroscopy analyte mutant

GSH

CDNB

GS-DNB

S-hexyl-GSH

A216KCou A216KCou/M208A A216KCou/M208S A216KCou/M208Y A216KCou/M208F A216KCou/M208G A216KCou/M208T A216KCou/M208L A216KCou/M208E

108 µM 179 µM 138 µM a 72 µM 99 µM 96 µM a 73 µM

1.3 mM 6.8 mM 1.2 mM a a 952 µM 775 µM 2.4 mM 601 µM

347 µM a a 128 µM a a a 46 µM a

2.5 µM a a a a a a a a

a

Not determined.

Figure 8. Close-up of the crystal structure (1GUH) of wt hGST A1-1 (A216 replaced with K216) in a complex with S-benzyl-GSH. Positions 216, 208, and 112 are shown in “stick” representation. The protein is dimeric but shown here as a monomer for reasons of clarity of presentation. The figure was done with the help of the PyMol (DeLano Scientific LLC) software.

Figure 7. Normalized fluorescence intensity at 408 nm was plotted against the concentration of free analyte, [analyte]free. The concentrations were calculated from Kd values. An equation that describes the dissociation of a bimolecular complex was fitted to the experimental data to obtain Kd values: S-hexyl-GSH (+, Kd ) 2.5 µM), GSH (4, Kd ) 108 µM), GS-DNB ([, Kd ) 347 µM), CDNB (1, Kd ) 1.3 mM). The solid lines represent the best fits to the experimental results.

respectively. Here, the negatively charged E208 and the positively charged K yield similar values, whereas R208 is an outsider. Is one of the proteins more sensitive than the others? Is one of the parameters more sensitive than the others? Upon inspection of Figure 4 (staple F-V and L-V) and Table 2, it is quite clear that the magnitude of changes reported from a protein depends on the ligand it encounters. For example, adding lithocholic acid to A216K/M208T gives rise to large changes in almost all parameters. On the other hand, this particular mutant does not signal any large differences between valeric

acid and fumaric acid monoethyl ester. One of the weakest candidates is of course A216K/M208C because it is prone to oxidation at the introduced C208 and the stability of this mutant is correspondingly less than that of the others (data not shown). However, from a practical point of view it is the total signature from the panel of proteins that is important, not the behavior of an individual protein. The related question is of course whether one of the parameters is more sensitive than the others. Again, judging from Figure 4 (staple F-V and L-V) and Table 2, it would appear that perhaps FRET data (∆FCou,FRET and ∆FCou,FRET,GSH) give rise to relatively large differences. But again, in this case less is not more, since a larger matrix allows for a higher degree of certainty. The ligands bind in a specific manner to the proteins. In a more quantitative part of the study, we performed titration studies with several molecules: GSH, CDNB, GS-DNB, and S-hexyl-GSH. We used a subset of double mutants and included the parent protein A216K as well. The experiments supplied us with information regarding the binding of the ligands (Kd values) and presented an opportunity to work with compounds that have absorbance spectra that interfere with the excitation and emission wavelengths of the probes. Even if it is not ideal,


Figure 9. Pictures from epifluorescence microscopy following immobilization of A216KCou to a PEG-based (hydrogel) surface: (A) sitespecific immobilization on a PDEA-activated hydrogel surface through C112 and (B) immobilization on an NHS-activated hydrogel surface through NHS-reactive residues on A216KCou.

the optical density problem can be dealt with using an equation that corrects the fluorescence intensity for inner filter effects (24). The titrations were performed in a black microplate with a clear bottom. Thus, we obtained information regarding the optical density, fluorescence intensity, and wavelength shift in the same experimental setup. The protein concentrations were 1 µM throughout the titrations, since we were expecting affinities in the low micromolar to low millimolar range, based on the Kd for GSH (190 ( 50 µM) of wild type hGST A1-1 (37). The results (Table 3) showed that the affinity for GSH for seven tested mutants varied over quite a narrow range (73–179 µM), in line with our expectations because GSH is known to bind in the G-site. The affinities for CDNB varied between 601 µM for A216K/M208E to 6.8 mM for A216K/M208A. The larger variation for CDNB binding between the mutants was not surprising because position 208 is located in the H-site, where CDNB is known to bind. The nucleophilic aromatic substitution reaction between GSH and CDNB to form GS-DNB is catalyzed by wt hGST A1-1, and the proteins showed affinities for GSDNB between those for GSH and CDNB, respectively. Also included in the affinity array was S-hexyl-GSH, which is a wellknown inhibitor of the R class GSTs. This ligand showed the highest affinity, 2.5 µM for A216K. The light-up effect was a positive surprise, enhancing the strength of the biosensor module (Figure 7). The coumarin-labeled protein scaffold can be anchored to a hydrogel microarray chip using C112 or nucleophilic residues. We used the by now well-studied protein A216Kcou to initiate the development of a surface-based biosensing system. The results from IR spectroscopy measurements (data not shown), MALDI-MS measurements following direct proteolytic digestion on the surface (Supporting Information), and fluorescence microscopy studies (Figure 9A), showed that A216Kcou could indeed be site-specifically introduced on the hydrogel surface through C112. We also found that A216Kcou could be attached to a NHS-activated hydrogel surface (Figure 9B), resulting in an even higher intensity of the emitted light,


indicating a higher concentration of A216Kcou in the spots. This is not surprising because the protein surface contains many potential NHS-reactive groups. We concluded that an optimization is needed before further development of the surface-based biosensor. Is this enough? This work constitutes “proof of principle” demonstrating that the ideas of the electronic nose and tongue can be transferred into the protein world. We have no illusions that this array of GST mutants could be used in real life to solve real life problems. Before the step from basic science to a real life application is taken, further diversification of the proteins is necessary as well as screening with real life analytes. However, it is very promising that ligands as diverse as n-valeric acid, fumaric acid monoethyl ester, lithocholic acid, CDNB, GSH, S-hexyl-GSH, and GS-DNB give rise to different signals that in more mature systems potentially can be distinguished and interpreted through pattern recognition. It is noteworthy that the Kd values we have measured (Table 3) span a range from low micromolar to low millimolar. The proposed use of our affinity array would in an initial stage be in a relatively controlled environment with a low noise from contributing compounds. This has so far also been true for the electronic nose sensor (38), which has for example been employed for quality measurements for commercial cardboard papers (39). However, the beauty of the GSTs lies in their promiscuity; it is easy to envision a selection where the scaffold is fine-tuned toward various types of analytes. Indeed, Mannervik et al. (40) have already performed a phage-display selection based on hGST A1-1. One can also envision a coupled system wherein GST(s), naturally existent, or mutant(s) attaches GSH to an analyte in situ to hitch a ride with the glutathione backbone and thus lower Kd values. This latter approach is of course only amenable if the analyte is an electrophile. Here also, if there is no GST that can catalyze the desired reaction, one can again think of a selection system to obtain the needed catalyst. The current system employs probes that are excited at relatively low wavelengths. This causes problems because many substances absorb light in this region and many biologically related solutions absorb or scatter light in this part of the spectrum. This obstacle can also almost certainly be overcome through some sort of selection or screening procedure to obtain mutants that can be labeled at K216 with a fluorescent acyl group that is excited at a higher wavelength. All of these things are made possible because, amazingly, hGST A1-1 seems to be an almost indestructible parent scaffold. It tolerates double mutations, rt handling over several days, and attachment to a surface without losing its structural integrity or ability to bind its natural substrates.

CONCLUSIONS We have constructed a multipurpose affinity array composed of a designed family of promiscuous proteins carrying introduced fluorescent residues. It is the pattern of changes generated by binding a specific ligand, i.e., its “signature” that constitutes the readout. This is an approach that turns the aspect of what constitutes a good biosensor upside-down. Here, the strength lies in the numbers; many relatively poor binders each contribute to the overall result.

ACKNOWLEDGMENT The authors express their sincere gratitude toward Professor Bengt-Harald Jonsson for his kind support. This work was financially supported by The Wenner-Gren Foundation, The Carl-Trygger Foundation, the Knut and Alice Wallenberg Foundation, and the Swedish Research Council.


Supporting Information Available: Stacked fluorescence spectra of the purified A216K/M208X mutants together with A216K and wt hGST A1-1, stacked fluorescence spectra of the coumarin-labeled A216K/M208X mutants together with A216KCou, specific activities toward CDNB and GSH, SDS–PAGE gels of the purified proteins, MALDI-MS spectra showing A216K in buffer before and after labeling with GSCCoubio, HPLC chromatograms showing reaction mixtures before and after treatment with NA beads, and direct proteolytic digestion (Staphylococcus aureus V-8) on the array surface. This material is available free of charge via the Internet at http://pubs.acs.org.

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