Design, Synthesis, and Evaluation of N- and C-Terminal Protein

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Design, synthesis and evaluation of N- and Cterminal protein bioconjugates as GPCR agonists Robert D Healey, Jonathan Wojciechowski, Ana Monserrat-Martinez, Susan L Tan, Christopher P Marquis, Emma Sierecki, Yann Gambin, Angela Monique Finch, and Pall Thordarson Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00716 • Publication Date (Web): 12 Jan 2018 Downloaded from http://pubs.acs.org on January 13, 2018

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Bioconjugate Chemistry

Design, synthesis and evaluation of NN- and CC-terminal terminal protein bioconbioconjugates as GPCR agonists Robert D. Healey#†§, Jonathan P. Wojciechowski#†, Ana Monserrat-Martinez‡§, Susan L. Tan†§, Christopher P. Marquis⊥, Emma Sierecki‡§, Yann Gambin‡§, Angela M. Finch§, Pall Thordarson†* †School

of Chemistry, the Australian Centre for Nanomedicine and the ARC Centre of Excellence in Convergent BioNano Science and Technology, §School of Medical Sciences, ‡EMBL Australia Node of Single Molecule Science, ⊥School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia #Author Contributions: Robert D. Healey and Jonathan P. Wojciechowski contributed equally to this work. ABSTRACT: A GPCR agonist protein, thaumatin, was site-specifically conjugated at the N- or C-terminus with a fluorophore for visualization of GPCR:agonist interactions. The N-terminus was specifically conjugated using a synthetic 2-pyridinecarboxyaldehyde reagent. The interaction profiles observed for N- and C-terminal conjugates were varied; Nterminal conjugates interacted very weakly with the GPCR of interest, whereas C-terminal conjugates bound to the receptor. These chemical biology tools allow interactions of therapeutic proteins:GPCR to be monitored and visualized. The methodology used for site-specific bioconjugation represents an advance in application of 2-pyridinecarboxyaldehydes for Nterminal specific bioconjugations.

INTRODUCTION The naturally occurring protein, thaumatin, is the sweetest tasting molecule known.1 The human tongue can detect the sweet taste of thaumatin in an aqueous solution as dilute as 50 nM.2 Thaumatin communicates its sweet tasting properties by interacting with a G protein-coupled receptor (GPCR), the sweet taste receptor.3 The sweet taste receptor is a class C GPCR, a heterodimeric receptor comprised of subunits Tas1R2 and Tas1R3.4,5 The sweet taste receptor is one of the best examples of a class C GPCR which can be activated by protein agonists, such as thaumatin, monellin, brazzein and lysozyme.6 Unlike typical small molecule agonists which activate class C GPCRs, such as glutamate,7 the mechanisms by which proteins agonists interact with class C GPCRs remains poorly characterized. Single molecule spectroscopy techniques have been shown to be powerful tools in studying the chemical biology of GPCRs.8 For fluorescence-based techniques, a limitation is the requirement of fluorescently labeled chemical probes and/or receptors. The site-selective chemical modification of native proteins remains a challenge for the synthesis of mono-functionalized bioconjugates, which are desirable for mechanistic studies.9 Moreover, the addition of a conjugated moiety onto a therapeutic protein can alter the physical properties of the protein, including binding affinity, binding kinetics and selectivity; considerations which must be taken into account when assessing the use of the bioconjugate as a chemical probe.10 Recently, a one-step native protein, N-terminal selective bioconjugation methodology was reported using functionalized 2pyridinecarboxyaldehydes (2PCA).11,12

In this work, we have prepared fluorescent thaumatin bioconjugates labeled at either C- or N-termini of thaumatin for interaction studies with a GPCR. We use a recombinantly engineered thaumatin with a C-terminal cysteine13 to prepare C-terminal fluorescent thaumatin bioconjugates (Fl-C-Thau) using thiol-maleimide Michael addition.14 Fluorescently labeled thaumatin bioconjugates at the N-terminus (Fl-N-Thau) are prepared using a synthetic 2-pyridinecarboxyaldehyde reagent, which is sitespecific for the N-terminal amine of native proteins.11 Using fluorescence spectroscopy and live cell microscopy, we show the N-terminal bioconjugates (Fl-N-Thau) bind less frequently with the sweet taste receptor than C-terminal bioconjugates (Fl-C-Thau). In addition, internalization of Fl-C-Thau was observed in FlpIn-293 cells through a pathway distinct from clathrin-mediated endocytosis, and in the absence of sweet taste receptors.

RESULTS AND DISCUSSION DISCUSSION Preparation of fluorescent thaumatin bioconjugates Fluorescent thaumatin bioconjugates were designed to minimize disturbance around critical surface residues for sweet-taste function (lysines and arginines).15–18 The structure of thaumatin is well documented (Figure 1), with the C-terminus located on the rear face of the protein when the structure is oriented to the active surface, presenting an ideal target for modification without loss of function (sweetness). In contrast, the N-terminus is located closer to the active surface hence thaumatin bioconjugates prepared by modification at the N-terminus may lose some activity.11 We sought to investigate both N- and C-terminus

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modification since retention of activity after bioconjugation can vary for each protein-probe bioconjugate.19,20

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tant concentrations improved yields to approximate 50% (Supplementary Figure 1). In other native protein bioconjugation strategies, decarboxylative photoredox of Cterminus native insulin was labeled in 49% yield.25 For proteins able to be produced in bacteria, and amenable to a site-directed mutation for an unnatural amino acid, a copper-free click approach may be utilized, reaching yields of 70% for zinc finger proteins26 and up to 80% for GST.27 Isolation of the bioconjugate, Fl-N-Thau and thaumatin from unreacted dye molecules was achieved through extensive dialysis and spin concentration against 50 mM MES buffer at pH 5.5. The low pH assisted with solubilization and stabilization of the thaumatin bioconjugate since thaumatin is more stable in acidic environments.24 After optimization, the N-terminal fluorescently labeled thaumatin, Fl-N-Thau, with 51% of the crude labeled product recovered, representing ≈ 5% overall yield.

Figure 1. A) Overview of thaumatin structure (PDB: 3ALD).18,21 B) Fluorophore used to label thaumatin. C) The bioconjugation strategies employed in this study to selectively label the C- and N-terminus.

N-terminal thaumatin bioconjugates (Fl-N-Thau) were prepared using a recently reported bioconjugation methodology.11,22 This method utilizes the selectivity of 2-pyridinecarboxaldehyde (2PCA) towards N-terminal amines, initially forming an imine, followed by cyclization via nucleophilic attack of the amide nitrogen on the peptide backbone to the electrophilic imine carbon. The cyclic imidazolidinone formed is stable in biological conditions whereas the imine is reversible and acid labile, hence irreversible side reactions with lysine residues or other amine groups are avoided due to the absence of a neighboring amide. This synthetic probe was modified with a rhodamine fluorophore (2PCA-Rho) to fluorescently label the Nterminus of thaumatin for later use in single molecule experiments. The reaction conditions for the conjugation of 2PCA-Rho to thaumatin were optimized by modifying the pH, temperature and concentration (Figure 2). The reaction was monitored using analytical HPLC-based size exclusion chromatography (HP-SEC); absorbance was monitored at 280 nm (protein) and 560 nm (rhodamine). Integrals of thaumatin retention peaks (tR = 4.81 min) were analyzed and the ratio of fluorophore/protein peak area was used to determine protein bioconjugation yield (Figure 2A and B). Reaction conditions were initially optimized using a constant ratio of thaumatin (10 µM solution) to 2PCA-Rho (1 mM, 100 equiv. to thaumatin). To investigate the effect of reaction temperature, a constant pH = 7.3 was used, with reactions performed at 4 °C, 22°C and 37 °C (Figure 2A). An increase in Fl-N-Thau conversion was observed at an elevated temperature (37 °C) with approximately 10% thaumatin labeled. Subsequently, reaction pH was optimized by monitoring reactions between pH 5.5 – 8.0 (Figure 2B). The reaction at pH 8.0 showed the highest product formation, however at this pH thaumatin exhibited reduced stability (Figure 2C).23,24 To ensure thaumatin stability, pH was maintained at 7.3 for all subsequent reactions and 10% methanol was included for solubility of 2PCA-Rho. Inclusion of methanol and an increase of reac-

Figure 2. Reaction condition optimization for the N-terminal labelling of thaumatin with 2PCA-Rho using HP-SEC. A) The effect of temperature on bioconjugation efficiency was monitored from the ratio of 560/280 nm absorbance. B) The effect of pH on bioconjugation efficiency. Overlay of HP-SEC chromatograms at 1 hour and 17 hours reaction times, monitored at C) 280 and D) 560 nm. In all reactions, [thaumatin] = 10 µM, [2PCA-Rho] = 1 mM. 2PCA-Rho shows two HP-SEC peaks due to the presence of regioisomers.

Site-specific C-terminal thaumatin-rhodamine bioconjugates were synthesized by using a recombinantly engineered thaumatin-FLAG-Cys (C-Thau) protein produced in yeast13, recombinant thaumatin produced in yeast has been shown to retain sweetness activity.17 This protein, CThau, contains a C-terminal cysteine separated from the protein by a FLAG tag, a peptide sequence commonly used for purification of recombinant proteins by affinity chromatography.28 Labelling was achieved using a commercially available rhodamine-maleimide (N,N,N’,N’-tetramethylrhodamine-5-maleimide) through the thiol-maleimide Michael addition.14 The reaction of CThau with rhodamine-maleimide was monitored using HP-SEC and the bioconjugate formation was confirmed through the co-elution of rhodamine (560 nm) absorbance with C-Thau (Supplementary Figure 2). Reduction of CThau prior to bioconjugation with tris(2-

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Bioconjugate Chemistry

carboxyethl)phosphine hydrochloride (TCEP·HCl) drastically improved the reaction efficiency.29 Fl-C-Thau bioconjugate was purified using a gravity flow PD-10 desalting column containing Sephadex G-25 resin to separate CThau and Fl-C-Thau from unreacted dye. The C-terminal bioconjugate, Fl-C-Thau was isolated in a modest 22% labeled yield. The yields of which are similar to those reported in the literature for cytochrome C Michael additions of cytochrome c in the presence of TCEP (27-50%)29,30 and BSA (25-50%).31,32

This suggests that bioconjugation of thaumatin at the C-terminus may prepare a dimeric species of rhodamine, induced by rhodamine stacking33 whereas the fluorophore of Fl-N-Thau remains in a monomeric form. Importantly, the emission spectra of Fl-C-Thau remains unchanged, which means the bioconjugate is still suitable for single molecule and confocal microscopy experiments. Bioconjugates were further characterized by LC-MS and CD (Supplementary Information). Interactions of bioconjugates with sweet taste receptor

Characterisation of fluorescent thaumatin bioconj bioconjuoconjugates The N- and C-terminal thaumatin bioconjugates were characterized by UV-Vis and SDS-PAGE (Figure 3). Fluorescent bands corresponding to labeled thaumatin bioconjugates (22 kDa) were observed using a rhodamine filter for Fl-N-Thau and Fl-C-Thau (Figure 3A). After Coomassie Blue staining, the proteins were confirmed for native thaumatin and recombinantly engineered C-Thau (Figure 3B).

Figure 3. Analysis of thaumatin bioconjugates Fl-N-Thau and Fl-C-Thau. A) Unstained SDS-PAGE imaged using a fluorescence reader (λex = 532 nm, emission filter: ≥ 575 nm). B) The same SDS-PAGE gel stained with Gel-Code blue protein stain and imaged with a LiCor Odyssey. C) UV-Vis (solid lines) and normalized fluorescence emission spectra (dashed lines) of thaumatin bioconjugates and unreacted dye probes. SDSPAGE gels were run under reducing conditions in the presence of 100 mM dithiothreitol.

The UV-Vis spectra (Figure 3C) of rhodamine-maleimide, Fl-N-Thau and 2PCA-Rho showed the same rhodamine absorbance maxima at 552 nm, consistent with the monomeric form of rhodamine.33 However, the absorption maxima of Fl-C-Thau was blue shifted to 523 nm (Figure 3C).

membranes Single-molecule spectroscopy experiments were performed to assess binding of the fluorescent thaumatin bioconjugates to over-expressed sweet taste receptors in mammalian AD-293 membrane fragments. Multicolor coincidence detection was used to determine co-localization between the eGFP-tagged sweet taste receptor (λem = 505545 nm) and N- or C-terminal bioconjugates, Fl-N-Thau and Fl-C-Thau, respectively (λem = 580 nm). Once the controls were set for a same concentration/number of photons registered for both bioconjugates, coincidence analyses were performed after incubating sweet taste receptor containing membrane preparations with the bioconjugates. In this assay, two excitation lasers (λex = 488 nm, eGFP and 561 nm, rhodamine) were focused within the same confocal volume, creating a very small observation volume. Brownian diffusion brings the fluorescent objects in and out of the confocal volume and the presence of either the receptor and/or the bioconjugate was detected by a burst of fluorescence in the eGFP or rhodamine channels, respectively. When bursts of photons happen simultaneously in both channels, binding between the receptor and bioconjugate was detected. The possibility of random co-diffusion in such a small volume is minimal.

Figure 4. A) Schematic of the single-molecule spectroscopy experiments between labeled Fl-C-Thau showing interactions with the GFP labeled sweet tasting GPCR-expressing receptor membranes. B) In contrast, Fl-N-Thau does not interact with the GPCR-expressing membranes. C) Selected 300 ms long time traces from the single molecule experiments with the signals from Fl-C-Thau (red) and eGFP-GPCR (green). Simultaneous burst in both traces (channels) indicate binding between the receptor and bioconjugate. D) Same as C) except the red traces correspond to Fl-N-Thau. See also Supplementary Figure 5 for the full time traces from these 2-minute long experiments.

The single-molecule spectroscopy experiments show a clear interaction between the C-terminal labeled thau-

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matin bioconjugate, Fl-C-Thau (Figure 4A and C) and the N-terminal bioconjugate – with the latter displaying minimal binding to the sweet taste receptor (Figure 4B and D). Longer intensity traces can be analyzed using the brightness method, as described recently.34 Here, we focus on the formation of multimers of the bioconjugates created by multiple binding to the same membrane were Fl-C-Thau shows larger and more frequent formation of oligomers in the photon counting histograms (Supplementary Figure 6). This experiment highlights that access to the Nterminus of thaumatin is important for the thaumatin:GPCR interaction, an observation consistent with previous studies which explored the importance of cationic charge on thaumatin activity.15 Although modification of the N-terminus of thaumatin has not been previously explored, modification of the C-terminus with pyridoxamine was reported to have no effect on the potency of thaumatin when analyzed by sensory analysis.18 The molecular mechanism of lingering sweet taste of thaumatin was investigated using the fluorescent thaumatin Fl-C-Thau. FlpIn-293 mammalian cells expressing the sweet taste receptor and FlpIn-293 cells with no sweet taste receptor were visualized using live cell confocal microscopy. Cells were incubated with Fl-C-Thau and endosomal marking protein then washed and imaged. In cells expressing sweet taste receptors, Fl-C-Thau bioconjugates were observed in distinct vesicles unstained by transferrin, and some transferrin-stained endosomes were found to contain Fl-C-Thau (Figure 5A).

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were found to interact with the plasma membrane and internalize into cells (Figure 5B). This sweet taste receptor-independent internalization process was via nonclathrin mediated internalization, as evident by a lack of co-localization with transferrin. In this work, we have prepared fluorescent thaumatin proteins as chemical biology tools for studying protein:GPCR interactions. We discovered that N-terminal bioconjugated thaumatin (Fl-N-Thau) does not interact as strongly with sweet taste receptors as C-terminal bioconjugated thaumatin (Fl-C-Thau). This further elucidates the interaction of thaumatin with the sweet taste receptor, and the importance of site-specific bioconjugation.15,18 The Cterminally labeled thaumatin bioconjugate, Fl-C-Thau show co-localization with the sweet taste receptor in single molecule experiments, so we utilized this for live cell imaging experiments. These experiments revealed that Fl-CThau internalizes into FlpIn-293 cells through a pathway distinct from clathrin-mediated endocytosis, and in the absence of sweet taste receptors. Also, some evidence of sweet taste receptor-mediated endocytosis through clathrin-mediated vesicles was detected, when Fl-C-Thau was applied to FlpIn-293 cells expressing the sweet taste receptor. We believe this work contributes to the understanding of interactions of protein agonists with class C GPCRs. In addition, this work highlights the importance of site-specific bioconjugation in mechanistic binding studies. This work has applications in the rational development of therapeutic protein agonists which bind GPCRs.

EXPERIMENTAL PROCEDURES PROCEDURES Materials and Methods Recombinant thaumatin-FLAG-cys (C-Thau) was expressed in the methylotrophic yeast Pichia pastoris as previously described13. Native plant thaumatin was obtained from Natex, UK and purified by strong cation exchange FPLC as described previously.36 N,N,N’,N’tetramethylrhodamine-5-maleimide and 5(6)-carboxyN,N,N’,N’-tetramethylrhodamine N-hydroxysuccinimidyl ester were purchased from Sigma Aldrich and used without further purification.

Synthesis

Figure 5. Live cell confocal microscopy showing internalization of Fl-C-Thau and transferrin-dylight649 when incubated with FlpIn-293 cells. A) FlpIn-293 cells expressing the sweet taste receptor, B) control FlpIn-293 cells not expressing the sweet taste receptor. Fl-C-Thau is displayed in green, transferrin-dylight649 is displayed in red. Arrows indicate internalised co-localised clathrin-mediated endocytotic vesicles containing Fl-C-Thau. Scale bar: 10 µm.

This data suggests that thaumatin may co-internalize with the sweet taste receptor, through clathrin-mediated endocytosis, as shown by co-localization with transferrin.35 However, the sweet taste receptor has not been reported to internalize before, so further molecular biology experiments should be performed to confirm this hypothesis. When FlpIn-293 cells not expressing the sweet taste receptor were stimulated with Fl-C-Thau, the bioconjugates

The synthesis of 2PCA-precursor, tert-butyl 4-((6-formylpyridin-2-yl)methyl)piperazine-1-carboxylate 3 has been previously described.11 Functionalization of 3 with rhodamine-NHS was achieved in two steps. Briefly, deprotection of 3 (2 mg, 6.6 µmol) was achieved using trifluoroacetic acid in dichloromethane, 1:1 (v/v) and stirred at 25 °C for 3 hours. The solvents were removed under reduced pressure. The resulting oil was redissolved in dichloromethane and concentrated under reduced pressure three times. Rhodamine-NHS (5 mg, 9.5 µmol), dissolved in anhydrous acetonitrile with triethylamine (33 µmol) was added and the reaction was stirred in the dark at 25 °C overnight, then purified using semi preparative-HPLC. Lyophilization of the eluate afforded 2PCA-Rho as a purple fluffy powder (1 mg, 1.6 µmol). Analytical HPLC: 96% (column: Grace Vision HT C18 HL, 2.1 x 50 mm, 3 µm; gradient: MeCN/0.1% aq formic acid: 0-5 min: 5:95, 5-20 min: 5:9520:80, 20-25 min: 5:95. tR = 13.40 min.). 1H NMR (600 MHz, d6-DMSO) δ 1.02-1.18 (4H, m) 3.03 (1H, d), 3.42-3.48 (2H,

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Bioconjugate Chemistry

m), 4.50 (1H, s), 6.61 (2H, d), 6.65 (2H, dd), 6.72 (1H, s), 6.73 (1H, d), 7.22 (1H, d), 7.67 (1H, dd), 7.76 (1H, d), 7.84 (1H, d), 7.90 (1H, s), 8.01 (1H, t), 9.93 (1H, s). 13C NMR (151 MHz, d6-DMSO) δ 5.45, 9.59, 29.25, 31.22, 47.72, 70.08, 88.40, 97.72, 111.36, 121.69, 125.38, 128.71, 130.21, 132.31, 139.07, 194.85. ESI-HR-MS m/z calcd for C36H36N5O5, 618.2711 [M+H]+; found 618.2705 [M+H]+.

FlFl-C-Thau bioconjugation Maleimide-thiol Michael additions were performed in 1.5 mL tubes and typical reaction volumes were 500 µL. Reactions were set up using stock solutions of thau-FLAGcys (1 mM), tris(2-carboxyethyl)phosphine (TCEP, 10 mM) and N,N,N’,N’-tetramethylrhodamine-5-maleimide (5 mM, in DMSO). Final protein concentration was typically 50100 µM. Prior to addition of maleimide, reduction was performed for at least 30 minutes using 1 molar equivalent of TCEP in Michael addition buffer (50 mM HEPES, 100 mM NaCl, 1 mM EDTA, pH = 6.8). Maleimide was then added to 5 molar equivalents per mole of protein and reactions incubated at room temperature for 2 hours. Protein was separated from unreacted dye molecules using PD-10 desalting columns (GE Life Sciences). Fractions containing only protein were pooled affording a mixture of unlabeled and labeled thaumatin bioconjugate, Fl-C-Thau. HPLC-MS (ESI) m/z 23.823 and MS (MALDI) m/z: 23,822 (calculated: 23,822).

FlFl-N-Thau bioconjugation N-terminal bioconjugates of native thaumatin were prepared using 2PCA-Rho as per previously reported conditions.11 Briefly, in 1.5 mL tubes, a stock solution of 2PCA-Rho was added in methanol/water and lyophilized. The residue was then dissolved to a final concentration of 10 mM using a solution containing 2PCA reaction buffer (50 mM HEPES, 100 mM NaCl, pH = 7.3), thaumatin at 50 µM concentration, and methanol 10% (v/v). The reaction was incubated at 37 °C for 16 hours then quenched by the addition of concentrated acidic buffer, MES buffer (500 mM, pH = 5.5). The entire mixture was repeatedly dialyzed against MES buffer (50 mM, pH = 5.5) using a 3.5 kDa MWCO dialysis membrane followed by spin concentrating and dialysis (10 kDa MWCO) against PBS buffer (pH = 7.4), affording the fluorescently labeled N-terminal thaumatin. Fl-N-Thau MS (MALDI) m/z: found 22,815. (calculated: 22,788).

High PerformancePerformance-Size Exclusion Chromatography (HP(HP-SEC) HP-SEC analysis was performed on an Agilent 1260 Infinity II Bio-inter HPLC system equipped with a Bio SEC-5, 150 Å, 4.6 x 300 mm column at a flow rate = 0.4 mL/min. The mobile phase consisted of an isocratic flow of 80 mM sodium phosphate, 120 mM NaCl, pH = 7.3 containing 20% (v/v) ethanol. Samples were injected using an autosampler with injection volumes of 10 µL.

SDS Page SDS-PAGE analysis of bioconjugates was performed typically with 1-3 µL of bioconjugation reaction mixed with 3 µL 4X LDS loading buffer (Life Technologies) and 6 µL MilliQ water. Samples were heated at 85 °C for 10 minutes

then loaded into a Bis-Tris 4-12% polyacrylamide gel and ran by electrophoresis at a constant 140 mA. After electrophoresis, the gel was fixed for 20 minutes in fixing solution (acetic acid/methanol/water, 10/50/40%, v/v/v), washed twice with milliQ water then imaged using a Typhoon FLA 9500 using the TAMRA filter (532 nm excitation laser, emission filter ≥575 nm. After fluorescent imaging, the gel was stained with Gel-Code blue (Thermo) and imaged using a LI-COR Odyssey.

Spectrophotometry UV-Vis and fluorescence spectra were recorded at 25 °C with UV-Vis (Cary 60) and fluorescence (Cary Eclipse) spectrophotometers, respectively. For absorbance measurements, the path length was 10 mm. Fluorescence measurements were recorded from 530 to 750 nm with excitation at 520 nm.

Single molecule fluorescence counting Single-molecule fluorescence counting experiments were performed on harvested AD-293 cell membranes constitutively expressing the sweet taste receptor, Tas1R2eGFP/Tas1R3, a heterodimeric G protein-coupled receptor.37 In this expression system, the C-terminal end of the Tas1R2 subunit was fused with eGFP. Cells were plated in 6-well dishes (1x106 cells/well) and incubated for 2 days in DMEM containing G418 and Hygromycin to retain stable expression of the receptor. Media was removed, and the cells were washed twice with PBS. Cells were harvested in 50 µL PBS by scraping then lysed on ice by passing through a fine gauge needle 50 times. Intact cells and cellular debris were removed by centrifugation at 500 g for 5 minutes. The resulting supernatant contained cell membrane fragments including eGFP-tagged sweet taste receptor. Single molecule fluorescence counting measurements were performed using freshly harvested membranes. Membranes were incubated with fluorescent thaumatin bioconjugates for 30 minutes at 37 °C, the concentration of membranes was the same in all experiments, and the concentration of bioconjugates was titrated down to a similar number of photons within the confocal volume. Samples were analyzed as previously described.38 Briefly, samples were pipetted into a custom-made silicone 192-well plate equipped with a 70 × 80 mm glass coverslip (ProSciTech, Australia). Plates were analyzed on a Zeiss LSM710 microscope with a Confocor3 module, at room temperature. Two lasers (488 nm and 561 nm) are co-focused in solution using a 40 × 1.2 NA water immersion objective (Zeiss, Germany); fluorescence was collected and split into GFPand rhodamine-channel by a 560 nm dichroic mirror. The GFP emission was further filtered by a 505–540 nm band pass filter and the rhodamine emission was filtered by a 580 nm long-pass filter.

Live cell microscopy Live cell microscopy experiments were performed on FlpIn-293 cells stably transfected with both subunits of the sweet taste receptor, Tas1R2 and Tas1R3, expression was inducible with tetracycline. The cells were plated onto poly-D-lysine coated coverslips and receptor expression was induced for 48 hours prior to imaging. Cells were washed once with HBSS (Hank’s balanced salt solution) containing 5.6 mM glucose and 1% BSA, w/v and placed on

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ice for 10 minutes to stop endocytosis. Coverslips were loaded into a POCmini-2 (PECON) perfusion chamber and the chamber was mounted into a 37 °C preheated Zeiss LSM 780 AxioObserver confocal laser scanning microscope equipped with a C-Apochromat 40x water immersion objective. A solution containing fluorescent thaumatin bioconjugate (Fl-C-Thau) at 10 µM and transferrin-dylight649 (Jackson ImmunoResearch, USA) at 25 µg/mL was flowed into the chamber. The cells were loaded with bioconjugate and transferrin for 2030 minutes then excess proteins were washed out using fresh buffer. Microscopy images were collected in the centre of the cell on the z-axis using the cell nucleus as reference. Image processing was performed using Fiji ImageJ software.39

ASSOCIATED ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.xxxxxxx. Additional details on Fl-NThau and Fl-C-Thau conjugations and characterization (PDF).

AUTHOR INFORMATION Corresponding Corresponding Author *E-mail: [email protected]. Phone: +61-(0)2-93854478.

Present Addresses Addresses †Robert D. Healey current address: Institut de Génomique Fonctionnelle, CNRS UMR-5203 INSERM U1191, University of Montpellier, 34094 Montpellier, France.

ORCID Robert D. Healey: 0000-0001-7025-0687 Jonathan P. Wojciechowski: 0000-0002-6272-515X Susan L. Tan: 0000-0002-6476-243X Christopher Marquis: 0000-003-1656-7215 Angela M. Finch: 0000-0002-4598-6745 Pall Thordarson: 0000-0002-1200-8814

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors thank the Mark Wainwright Analytical Centre (UNSW) for access to instruments and protein sequencing services. The authors also acknowledge the UNSW Recombinant Products Facility for access to instruments and expertise in recombinant thaumatin expression and purification. We acknowledge the Australian Research Council for an ARC Centre of Excellence grant (CE140100036) to PT. Support for this project was also provided by Neptune Bio-Innovations Pty. Ltd. to RDH and PT. We thank the Australian Government for PhD scholarships to RDH, JPW and SLT.

REFERENCES (1) van der Wel, H., and Loeve, K. (1972) Isolation and characterization of thaumatin I and II, the sweet-tasting proteins from Thaumatococcus daniellii Benth. Eur. J. Biochem. 31, 221–225. (2) Ide, N., Sato, E., Ohta, K., Masuda, T., and Kitabatake, N. (2009) Interactions of the sweet-tasting proteins thaumatin and lysozyme with the human sweet-taste receptor. J. Agric. Food Chem. 57, 5884– 90.

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(27) Jang, S., Sachin, K., Lee, H., Kim, D. W., and Lee, H. S. (2012) Development of a Simple Method for Protein Conjugation by CopperFree Click Reaction and Its Application to Antibody-Free Western Blot Analysis. Bioconjug. Chem. 23, 2256–2261. (28) Hopp, T. P., Prickett, K. S., Price, V. L., Libby, R. T., March, C. J., Pat Cerretti, D., Urdal, D. L., and Conlon, P. J. (1988) A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio/Technology 6, 1204–1210. (29) Hvasanov, D., Mason, A. F., Goldstein, D. C., Bhadbhade, M., and Thordarson, P. (2013) Optimising the synthesis, polymer membrane encapsulation and photoreduction performance of Ru(ii)- and Ir(iii)bis(terpyridine) cytochrome c bioconjugates. Org. Biomol. Chem. 11, 4602. (30) Peterson, J. R., Smith, T. A, and Thordarson, P. (2010) Synthesis and room temperature photo-induced electron transfer in biologically active bis(terpyridine)ruthenium(II)-cytochrome c bioconjugates and the effect of solvents on the bioconjugation of cytochrome c. Org. Biomol. Chem. 8, 151–162. (31) Hvasanov, D., Nam, E. V., Peterson, J. R., Pornsaksit, D., Wiedenmann, J., Marquis, C. P., and Thordarson, P. (2014) One-pot synthesis of high molecular weight synthetic heteroprotein dimers driven by charge complementarity electrostatic interactions. J. Org. Chem. 79, 9594–9602. (32) Riener, C. K., Kada, G., and Gruber, H. J. (2002) Quick measurement of protein sulfhydryls with Ellman’s reagent and with 4,4′-dithiodipyridine. Anal. Bioanal. Chem. 373, 266–276. (33) Selwyn, J. E., and Steinfeld, J. I. (1972) Aggregation of equilibriums of xanthene dyes. J. Phys. Chem. 76, 762–774. (34) Gambin, Y., Giles, N., O’Carroll, A., Polinkovsky, M., Hunter, D., and Sierecki, E. (2017) Single-molecule fluorescence reveals the oligomerisation and folding steps driving the prion-like behaviour of ASC. J. Mol. Biol. DOI: 10.1016/j.jmb.2017.12.013. (35) Dale, L. B., Bhattacharya, M., Seachrist, J. L., Anborgh, P. H., and Ferguson, S. S. (2001) Agonist-stimulated and tonic internalization of metabotropic glutamate receptor 1a in human embryonic kidney 293 cells: agonist-stimulated endocytosis is beta-arrestin1 isoformspecific. Mol. Pharmacol. 60, 1243–1253. (36) Healey, R. D., Prasad, S., Rajendram, V., and Thordarson, P. (2015) Unravelling the interaction between α-cyclodextrin with the thaumatin protein and a peptide mimic. Supramol. Chem. 27, 414–419. (37) Jiang, P., Ji, Q., Liu, Z., Snyder, L. a, Benard, L. M. J., Margolskee, R. F., and Max, M. (2004) The cysteine-rich region of T1R3 determines responses to intensely sweet proteins. J. Biol. Chem. 279, 45068–75. (38) Sierecki, E., Giles, N., Bowden, Q., Polinkovsky, M. E., Steinbeck, J., Arrioti, N., Rahman, D., Bhumkar, A., Nicovich, P. R., Ross, et al. (2016) Nanomolar oligomerization and selective co-aggregation of αsynuclein pathogenic mutants revealed by single-molecule fluorescence. Sci. Rep. 6, 37630. (39) Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, et al. (2012) Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682.

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TOC graphic 40x17mm (300 x 300 DPI)

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Figure 1. A) Overview of thaumatin structure (PDB: 3ALD).18,21 B) Fluorophore used to label thaumatin. C) The bioconjugation strategies employed in this study to selectively label the C- and N-terminus. 66x52mm (300 x 300 DPI)

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Figure 2. Reaction condition optimization for the N terminal labelling of thaumatin with 2PCA-Rho using HP-SEC. A) The effect of temperature on bioconjugation efficiency was moni-tored from the ratio of 560/280 nm absorbance. B) The effect of pH on bioconjugation efficiency. Overlay of HP-SEC chromatograms at 1 hour and 17 hours reaction times, monitored at C) 280 and D) 560 nm. In all reactions, [thaumatin] = 10 µM, [2PCA Rho] = 1 mM. 2PCA-Rho shows two HP-SEC peaks due the presence of regioisomers. 76x68mm (300 x 300 DPI)

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Figure 3. Analysis of thaumatin bioconjugates Fl-N-Thau and Fl-C-Thau. A) Unstained SDS-PAGE imaged using a fluorescence reader (λex = 532 nm, emission filter: ≥ 575 nm). B) The same SDS-PAGE gel stained with Gel-Code blue protein stain and imaged with a LiCor Odyssey. C) UV Vis and normalized fluorescence emission spectra of thaumatin bioconjugates and unreacted dye probes. SDS-PAGE gels were run under reducing conditions in the presence of 100 mM dithiothreitol. 103x136mm (300 x 300 DPI)

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Figure 4. A) Schematic of the single-molecule spectroscopy experiments between labelled Fl-C-Thau showing interactions with the GFP labelled sweet tasting GPCR-expressing receptor membranes. B) In contrast, Fl-N-Thau does not interact with the GPCR-expressing membranes. C) Selected 300 ms long time traces from the single molecule experiments with the signals from Fl-C-Thau (red) and eGFP-GPCR (green). Simultaneous burst in both traces (channels) indicate binding between the receptor and bioconjugate. D) Same as C) except the red traces correspond to Fl-N-Thau. See also Supplementary Figure 5 for the full time traces from these 2-minute long experiments. 46x31mm (300 x 300 DPI)

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Figure 5. Live cell confocal microscopy showing internalization of Fl-C-Thau and transferrin dylight649 when incubated with FlpIn-293 cells. A) FlpIn-293 cells expressing the sweet taste receptor, B) control FlpIn-293 cells not expressing the sweet taste receptor. Fl-C-Thau is displayed in green, transferrin dylight649 is displayed in red. Arrows indicate internalised co-localised clathrin-mediated endocytotic vesicles containing Fl-C-Thau. Scale bar: 10 µm. 84x67mm (300 x 300 DPI)

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