Green Fluorescent Probe for Imaging His6-Tagged Proteins Inside

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Green fluorescent probe for imaging His-tagged proteins inside living cells Ya Yang, Nan Jiang, Yau-Tsz Lai, Yuen-Yan Chang, Xinming Yang, Hongzhe Sun, and Hongyan Li ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01128 • Publication Date (Web): 23 Apr 2019 Downloaded from http://pubs.acs.org on April 23, 2019

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Green fluorescent probe for imaging His6-tagged proteins inside living cells Ya Yang, Nan Jiang, Yau-Tsz Lai, Yuen-Yan Chang, Xinming Yang, Hongzhe Sun* and Hongyan Li * Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, China KEYWORDS: Small fluorescent probe, photoactivation, His6-tagged protein, live cell imaging, membrane permeability

ABSTRACT: Small molecule-based fluorescent probes offer great opportunities for specifically tracking proteins in living systems with minimal perturbation on the protein function and localization. Herein, we report a small green fluorescent probe (Ni2+-NTA-AF) consisting of a Ni2+-NTA moiety, a fluorescein and an arylazide group, binds specifically to His6-tagged proteins with fluorescence enhancement in vitro upon photoactivation of the arylazide group. Importantly, the probe can cross the cell membranes and stoichiometrically label His6-tagged proteins rapidly (~15 mins) in living prokaryotic and eukaryotic cells exemplified by a DNA repair protein Xeroderma pigmentosum group A (XPA). Using the probe, we successfully visualized Sirtuin 5, which is localized to the mitochondria. This probe exhibits high quantum yields and improved solubility, offering a new opportunity for imaging intracellular His6-tagged proteins inside living cells with better contrast.

Fluorescent imaging has long been used as an indispensable and ubiquitous technique for real-time monitoring intracellular events with high spatial resolution1. Protein labelling with fluorescent molecules facilitates the protein visualization and functional studies2. Genetically encoded fluorescent proteins have the advantages to be transiently transfected and genetically incorporated into the proteins of interest (POI) without chemical synthesis3, but their relative bulky structures might perturb the localization and functions of POIs4-5. As a consequence, short peptide-based tag systems have been widely employed to selectively label proteins in vivo to minimize the perturbation6-7. Recently, small molecule-based fluorescent probes have been developed to selectively and specifically recognize the peptide sequence of the POIs in situ to permit good structural preservation8-9. Traditional fluorescent labelling needs fluorescence bleaching or quenching to reduce the background noise, whereas this may damage proteins in cells10. Thus, photoactivable fluorescence turn-on probes serve as an ideal alternative for protein labelling11-13. Generally, the probes should be small, biostable, easily synthesized and importantly, it should be nonfluorescent until a brief pulse or a simple activating reaction 14. FlAsH and ReAsH are typical small molecule-based fluorescent probes to label genetically tetracysteine-tag fused proteins15-16. However, 1,2-ethanedithiol (EDT) must be applied to reduce the background noise and enhance the labeling. Moreover, it is sensitive to the intracellular redox environment due to the reduced form of tetracysteine motif. This system was also reported to non-specifically bind to similar cysteine-rich proteins, leading to unspecific labeling and background problems17. The utility of the hexahistidine-nickel-nitrilotriacetate (His6Ni2+-NTA) system for the purification of His6-containing proteins was reported 30 years ago18. Nowadays such a system has been widely used as a valuable tool for the affinity purification of recombinant proteins with His6-tag genetically engineered to the N- or Cterminus19. The first NTA-based fluorescent reagent was reported in 2001 for the detection of His-tagged proteins20. Subsequently, this type of probe was utilized to in vivo selectively label membrane His-tagged proteins21. To date, various Ni2+-NTA based fluorescent probes have been made by conjugating one or more NTA

moieties with a fluorphore17, 22-25, each with unique utility for both in vitro and in vivo labelling of His6-tagged proteins. Unfortunately, nearly all NTA based probes were unable to track His6-tagged and His10-tagged proteins inside living cells unless cell-penetrating peptides (CPPs) were conjugated with the probe to facilitate its crossing of the cell membrane24-26.

Scheme 1. Schematic illustration of selective labelling and identifying His6-tagged protein using Ni2+-NTA-AF

Recently, we have developed a membrane permeable coumarin based fluorescent probe Ni-NTA-AC, allowing rapid and specific labelling of His6-tagged proteins in mammalian cells and plant tissues with significant fluorescence turn-on27. However, its quantum yield (QY) is low and the imaging visual contrast is far from satisfactory. Moreover, the blue wavelength is not ideal because of poor tissue penetrance and high levels of autofluorescence from cells. To circumvent these issues, a red derivative Ni-NTA-AB was synthesized to label His6-tagged proteins but the labelling was achieved only in bacterial cells with the aid of Tween 80 because of its poor solubility28. Herein, we report a fluorescein-based small fluorescent probe, Ni2+-NTA-AF, consisting of an arylazide, a fluorescein and a Ni2+NTA moiety. The probe binds specifically to His6-tagged proteins, leading to over 15-fold fluorescence enhancement upon UV activation. Importantly, the probe could rapidly enter the bacteria and cross the membranes of different cellular organelles with high selectivity to image His6-tagged proteins. Upon UV exposure, the arylazide group was activated to stimulate the fluorescence enhancement and form irreversible labelling to the targeted proteins (Scheme 1). This green probe with higher QY and better solubility

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should offer great opportunity to image proteins inside living cells with better contrast.

RESULTS AND DISCUSSION Design, synthesis and characterization of Ni2+-NTA-AF. The previously reported fluorescent probes based on His6-Ni2+NTA system suffered from poor membrane permeability due to their relatively big sizes or excessive negative charges, limiting their applications to image only the membrane proteins24-26. We recently designed cell-permeable fluorescent probes Ni-NTA-AC and Ni-NTA-AB, which achieved successful labelling of His6-tagged proteins inside live cells27-28. To reduce the phototoxicity from the blue laser, eliminate blue autofluorescence from cells, as well as to improve the solubility and cellular uptake of the probe, we therefore design a green fluorescent probe, i.e. Ni2+-NTA-AF. Fluorescein was selected as the green fluorophore because of its high fluorescence QY and longer excitation and emission wavelength29. Ni2+-NTA moiety was designed for specifically targeting the His6tag of a protein, and a small sized non-toxic arylazide group was introduced for its capability to form an irreversible covalent bond with the labelled proteins upon UV activation, thus enhancing the anchoring to the POIs in addition to the moderate binding of Ni2+ with His6-tag30. Moreover, incorporation of arylazide may lead to the photoactivatable fluorescence turn-on owing to the spectroscopic properties of fluorescein31-33.

Scheme 2. Synthetic scheme of ligand NTA-AF

The ligand NTA-AF was synthesized with a four-step reaction (Scheme 2 and SI). Commercially available fluoresceinamine was diazotized to form azidofluorescein (Compound 1) with sodium azide under acidic conditions. NTA methyl esters (Compound 2) were synthesized with methanol and thionyl chloride to protect the carboxyl groups during the subsequent condensation reaction. A protected amide (Compound 3) was obtained with EDC and HOBT as the coupling reagents and purified by silica gel column. The final ligand NTA-AF was obtained by the hydrolysis with lithium hydroxide. The identity and purity of NTA-AF were confirmed by ESI-MS and NMR spectroscopy (Figure S1-S4). The probe Ni2+NTA-AF was prepared by mixing equimolar amounts of NTA-AF with Ni2+ (as NiSO4) in water or buffered aqueous solution for at least 30 minutes at room temperature in the dark. The successful formation of 1:1 complex was confirmed by ESI-MS, which gave rise to one peak at m/z 672.1, corresponding to the calculated m/z value of 672.1 (Figure S5). The probe Ni2+-NTA-AF exhibited maximum excitation at 494 nm and emission at 514 nm (Figure S6) with QY of 0.66, which was determined with fluorescein in 0.1 M NaOH (QYst = 0.92) as the standard according to reported method34. However, in neutral condition, the probe exhibits very low fluorescence. It showed excellent stability before photoactivation as no photobleaching was observed upon continuous scanning (Figure S7).

Evaluation of Ni2+-NTA-AF for labelling His6-tagged proteins in vitro. The binding of Ni2+-NTA-AF towards proteins was

first examined in solution. Xeroderma pigmentosum group A35 without (XPA) and with (His-XPA) genetically fused His6-tag at its N-terminus were used as a showcase study. The proteins were overexpressed and purified with the same methods as described previously27. The binding between His-XPA and the probe was first examined by MALDI-TOF MS. 10 µM of His-XPA were incubated with 0, 1, 2, and 5 molar equivalents (0, 10, 20, 50 µM) of Ni 2+NTA-AF or 5 molar equivalents (50 µM) of NTA-AF on ice for 30 minutes, then the mixtures were activated with or without 365 nm UV for 20 minutes prior to analysis. As shown in Figure 1A (c and d), incubation of His-XPA with Ni2+-NTA-AF upon UV exposure resulted in two new peaks at m/z 33419 and 34080, which were assigned to be His-XPA with one and two Ni2+-NTA-AF bound, respectively. In contrast, no such peaks were observed on the MS spectra and almost no green fluorescent bands can be detected when His-XPA was incubated with even 5 molar equivalents of ligand NTA-AF (Figure S8), indicating that Ni2+ ion is essential for successful binding of the protein by the probe. To further demonstrate the binding between the probe and HisXPA, Isothermal titration calorimetry (ITC) was conducted. The dissociation constant was determined to be 6.8 ± 0.6 μM (Figure S9), in consistence with the moderate binding between Ni2+-NTA and His6-tag30, 36, but such a moderate binding affinity may result in the failure of the probe’s accumulation near the POIs. To overcome such an issue, fluorophores conjugating to di-, tri- and tetraNTA have been previously reported22, 24-26, 37, unfortunately, these probes without CPPs attached suffer from poor membrane permeability owing to their large sizes and/or highly negative charges. Inspired by our previous design27-28, an arylazide group was incorporated into the 5-carbon position of fluorescein to provide strong covalent binding between the probe and the POIs upon UV activation. To further confirm the strong binding after UV exposure, SDSPAGE analysis was then conducted. A series of samples containing either 5 μM His-XPA or XPA, 5 μM Ni2+-NTA-AF or NTA-AF, in the presence or absence of 250 μM of EDTA were prepared. The samples were then divided into two parts: one with and the other without UV irradiation at 365 nm for 20 minutes prior to SDSPAGE analysis. As shown in Figure 1B, an intense green fluorescent band could only be observed on the gel for His-XPA upon mixing with Ni2+-NTA-AF irradiated by UV. In contrast, no obvious green fluorescence was detected under identical conditions when XPA was used, or when the samples were prepared without UV irradiation. Interestingly, in the presence of 50 equivalents of EDTA, no observable green fluorescent band was found for HisXPA when incubating with Ni2+-NTA-AF, possibly owing to the removal of Ni2+ from the probe by EDTA. This is in accordance with the observation that no new peak was generated from MALDIMS when incubating His-XPA with NTA-AF irradiated by UV (Figure 1A, b). Next, we examined the fluorescence response of the probe towards proteins by fluorescence spectrophotometry. Incubation of 10 molar equivalents of His-XPA or XPA with Ni2+-NTA-AF (5 μM) led to negligible changes on the fluorescence. Upon exposure to UV irradiation at 365 nm by a UV lamp (18000 µW/cm2 at 15 inches), the fluorescence increased with the irradiation time and reached a plateau at about 15 minutes, whereas only ca. 6.0-fold and 4.8-fold fluorescence enhancement was observed in the presence of His-XPA and XPA respectively (Figure S10, Insert). Fluorescein is an ideal core for the construction of the OFF-ON fluorescent molecules considering its non-fluorescent closed spirolactam form and the highly fluorescent open quinoid form32. UV irradiation resulted in an observable fluorescence turn-on, at the same time the color of the solution changed from colorless to deep yellow (Figure S11)32. This is likely due to the fact that UV exposure acti-

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ACS Sensors vates arylazide, resulting in a highly fluorescent form of the fluorophore; 31, 33, 38 upon binding to a His-tagged protein, covalent bonds were formed between the probe and the POIs, forming a larger conjugation system, which is evidenced by ~10 nm red shift of fluorescence emission (Figure S10). In addition, hydrophobic interactions between the probe and labeled protein might further enhance the fluorescence. The lower (6-fold) fluorescent enhancement is likely due to the lower energy generated by the UV light, which could not fully convert the probe into highly fluorescent open form. This is supported by our data that fold of fluorescence enhancement is dependent on the power of the UV lamp (Figure S12).

Figure 1. (A) The MALDI-TOF MS spectra of His-XPA in the absence and presence of Ni2+-NTA-AF or NTA-AF. a: His-XPA + UV; b: His-XPA + 5 eqv. NTA-AF + UV; c: His-XPA + 1 eqv. Ni2+NTA-AF + UV; d: His-XPA + 2 eqv. Ni2+-NTA-AF + UV; e: HisXPA + 5 eqv. Ni2+-NTA-AF + UV. The arrows referred to the peaks of His-XPA (m/z = 32747, calculated 32742) and His-XPA combined with one or two Ni2+-NTA-AF respectively (m/z = 33419, 34080). (B) The selectivity in labelling His6-tagged proteins in vitro confirmed with SDS-PAGE under different conditions. (C) The fluorescence increase of 5 μM Ni2+-NTA-AF incubated HisXPA upon UV activation at different time intervals. To confirm the formation of protein-probe adducts via covalent bonds upon UV exposure, SDS-PAGE analysis was used. Ni2+NTA-AF (5 μM) incubated with 5 molar equivalents of His-XPA was irradiated by a UV lamp (18000 µW/cm2 at 15 inches) for different time intervals (0, 0.17, 0.5, 1, 2, 5, 10, 15, 20, 30 min), then subjected to SDS-PAGE and fluorescence imaging. We found that the fluorescence intensities increased with time and reached a plateau at about 15 minutes, where ~ 15-fold increase in the fluorescence referred to that of 0.17 min was observed. However, no fluorescence was observed without UV exposure under identical conditions. Taken together, we have developed a green photoactivatable fluorescence turn-on probe, Ni2+-NTA-AF, which selectively binds to the His6-tag of a protein via Ni2+ ion, and the binding was significantly strengthened upon photoactivation of the arylazide due to the formation of covalent bonds between the probe and the labelled proteins. The labelling yield of Ni2+-NTA-AF to His-XPA was monitored by incubating 5 μM His-XPA with increasing amounts (0, 0.2, 0.5, 1, 2, 3, 5, 10, 15 and 20 equivalents) of Ni2+-NTA-AF, then UV irradiation at 365 nm for 20 minutes prior to SDS-PAGE. As shown in Figure 2A (bottom), about 25% of His-XPA was labelled in the presence of 1 molar equivalent of Ni2+-NTA-AF, and the labelling

yield reached over 80% when 10 molar equivalents of probe were present.

Figure 2. (A) Labelling efficiency of Ni2+-NTA-AF to 5 μM HisXPA monitored by Coomassie Blue and fluorescence staining (up), and the corresponding normalized fluorescence intensity changes were plotted against the equivalent ratios (bottom). (B) The effects of GSH on the labelling between Ni2+-NTA-AF and 5 μM His-XPA monitored by Coomassie Blue and fluorescence staining (up), and the corresponding normalized fluorescence intensity changes were plotted against the GSH concentration (bottom). (C) Sensitivity of Ni2+-NTA-AF on labelling His-tagged protein. The intensities of green fluorescence bands on SDS-PAGE were compared with those of His-antibody Western blot (up). The integrated fluorescence intensity of the fluorescent bands showed the linear range (R2=0.9946) for fluorescence labelling of His-XPA by Ni2+-NTA-AF (bottom). To examine the potential effects of biological reducing agents on protein labelling, we selected the most abundant intracellular reductant glutathione (GSH) as a showcase study. Gradient amounts of GSH (0, 0.02, 0.05, 0.2, 1, 2, 5, 10 mM) were supplied to the mixture of 5 μM His-XPA and Ni2+-NTA-AF. The samples were then UV irradiated prior to SDS-PAGE. As shown in Figure 2B, all the green fluorescent bands on the SDS gel remained almost the same (Figure 2B, up) and based on the signals intensities quantified by ImageJ, over 80% fluorescence intensity were observable even in the presence of up to 10 mM GSH (Figure 2B, bottom), suggesting that GSH has insignificant effects on the protein labelling process. Similarly, Cysteine (Cys) and Homocysteine (Hcy) had no obvious effect on the His-tagged protein labelling process by the probe (Figure S13). The sensitivity of Ni2+-NTA-AF on labelling of His6-tagged proteins was then evaluated by comparing with Western blot using anti-His6-Tag antibody. A series of concentrations (2.5, 25, 50, 100, 200, 400, 600, 800 and 1000 ng) of His-XPA were incubated with 10 molar equivalents of Ni2+-NTA-AF, then subjected to UV irradiation at 365 nm for 20 minutes prior to SDS-PAGE and fluorescence imaging. The integrated fluorescence intensity on the vertical direction with Image J gave rise to the detection limit, green bands with signal-to-noise (S/N) ratio ≥ 2 were regarded as detectable. As shown in Figure 2C, the fluorescence intensity increased with the increase in protein concentration, and the detection limit was estimated to be around 25 ng, which is comparable to that obtained from Western blot. However, a wider range of linearity (25-1000 ng examined, R2=0.9946) between the fluorescence intensities and the amounts of proteins labelled by Ni2+-NTA-AF was noticeable since a linear range from 25 to 200 ng was generally found when

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using Western blot to quantify proteins concentration39. As a consequence, Ni2+-NTA-AF also serves as a promising agent to quantify His6-tagged proteins.

Imaging of His-XPA in live E. coli cells. We then examined the feasibility of Ni2+-NTA-AF in labelling His6-tagged proteins in bacterial cells. 25 µM Ni2+-NTA-AF were incubated with E. coli cells expressing His-XPA or XPA and E. coli cells with mock vector transformed. The cells were then washed with HBSS for three times and treated with propidium iodide (PI) before confocal imaging to examine the cell viability and membrane integrity. Confocal images were taken before and after UV (405 nm blue channel equipped in the confocal microscope for several seconds due to its high energy27) irradiation. As shown in Figure 3A, only E. coli cells that overexpressed His-XPA exhibited intense green fluorescence after UV exposure, and subsequent analysis on the cell lysates showed only one bright green fluorescent band with a molecular weight of about 32 kDa (calculated MW 32.7 kDa for His-XPA) on the SDS gel (Lane 1, Figure S14), indicating that the probe selectively binds to His-XPA and the photoactivation resulted in structural rearrangement of the probe and the formation of covalent bond between the protein and the probe, leading to a larger conjugation system. In contrast, E. coli cells with XPA overexpressed or with mock vector exhibited very low fluorescence, which contributed to the background signal. Also, few cells were stained red by PI, suggesting that most of the cells were alive (93.74% cell viability for His-XPA E. coli group, 94.87% for XPA E. coli group and 95.36% for mock vector E. coli group). In comparison with our blue probe Ni2+-NTA-AC, Ni2+-NTA-AF exhibits a better contrast (11.40±3.78 vs 4.38±2.24), estimated by dividing the fluorescence of treated group by the fluorescence of the background noise (unstained group) (Figure S15). Taken together, we show that Ni2+NTA-AF could enter live E. coli cells and selectively label His-XPA with fluorescence turn-on. Visualization of His-XPA in live mammalian cells. To demonstrate the capability of Ni2+-NTA-AF in labelling His6-tagged proteins in live mammalian cells, we first evaluated the potential toxicity of Ni2+-NTA-AF on a mammalian cell line by MTT assay. Supplementation of Ni2+-NTA-AF at concentrations up to 50 µM Ni2+-NTA-AF to COS-7 cells led to no obvious effects on cell viability (Figure S16), indicative of negligible toxicity of the probe. Next we examined whether the probe could enter mammalian cells. Confocal imaging of COS-7 cells treated with 25 µM Ni2+-NTA-AF at different incubation time intervals was conducted. The green fluorescence appeared in the cytosol in 5 minutes and gradually increased throughout the cell in 15 minutes. The quantified intensities also showed that Ni2+-NTA-AF was able to keep steady in ca. 15 minutes (Figure S17). In contrast, 25 µM NTA-AF gave rise to only very low fluorescence inside COS-7 cells after incubating even for 1 hour (Figure S18), suggesting that Ni2+ ion is necessary for the membrane permeability of the probe. This is likely due to the fact that the overall negative charge of Ni2+-NTA-AF was reduced in comparison to NTA-AF. Moreover, Ni2+-NTA-AF might share a similar sandwich-like structure to the blue and red probes owning to the interactions between Ni2+-NTA and the fluorophore π system40, allowing entry of the probes into cells. Subsequently, the genetically fused His-XPA plasmids were transiently expressed in COS-7 cells and 25 µM Ni2+-NTA-AF were supplied to the cells, and cells were then washed, UV irradiated with 405 nm blue channel equipped in the confocal microscope for 1 minute prior to confocal imaging. Green fluorescence was observed mainly at the nucleus region of the transfected cells, where XPA protein expressed35 (Figure 3B). To further confirm identity of the protein, the nucleus of the cells with or without transfection after treatment with Ni2+-NTA-AF and UV exposure were extracted for SDSPAGE and fluorescence imaging. The green fluorescent band was observed only in the nuclei of His-XPA transfected COS-7 cells

with Ni2+-NTA-AF treatment. By using Western blot, the purified His-XPA protein with the same treatment gave rise to a green band at a similar molecular weight (Figure 3C), confirming that the labelled protein is indeed His-XPA expressed in the cells. Therefore, we conclude that Ni2+-NTA-AF can be utilized to track intracellular His6-tagged proteins in mammalian cells.

Figure 3. (A) Confocal imaging of mock vector, XPA and HisXPA overexpressed E. coli cells with 25 µM Ni2+-NTA-AF. Only His-XPA overexpressed E. coli cells incubated with Ni2+-NTA-AF showed green fluorescence, and green fluorescence only lighted up after subjecting with 405 nm UV laser for a few seconds. n≥5, scale bar: 5 µm. (B) The confocal imaging of protein labelling in transfected COS-7 cells incubated with 25 µM Ni2+-NTA-AF and irradiated with UV laser. The green fluorescence was found in the nuclear region, where His-XPA expressed. Scale bar: 10 µm. (C) The corresponding fluorescence SDS-PAGE and Western blot analysis of cell lysates as used in confocal imaging in B, all the samples were UV irradiated before gels. Lane 1: nuclear extraction of COS-7 cells with 25 µM Ni2+-NTA-AF; Lane 2: nuclear extraction of His-XPA transfected COS-7 cells; Lane 3: nuclear extraction of His-XPA transfected COS-7 cells with 25 µM Ni2+-NTAAF; Lane 4: 0.5 µM purified His-XPA protein incubated with Ni2+NTA-AF.

Fluorescent labelling of His-SIRT5 in mammalian cells. SIRT5 is a protein belonging to Sirtuin 2 family and is a NADdependent demalonylase and desuccinylase mainly localized to the mitochondria41. The protein is known to facilitate cancer cell growth and drug resistance in non-small cell lung cancer. The His6SIRT5 plasmid was constructed with His6-tag genetically fused into its C terminus, and transfected into HeLa cells. HeLa cells with or without His-SIRT5 transfection were incubated with 25 μM Ni2+-NTA-AF in HBSS for 30 minutes. After washing, the treated cells were subjected to 405 nm blue channel equipped in the confocal microscope for 1 minute. As shown in Figure 4A, green fluorescence was observed mainly in the mitochondria of transfected cells but not in the cells without His6-SIRT5 transfection. The overlap coefficient for co-localization was calculated to be 0.75 with ZEN software. To further confirm the identity of labelled protein, cells with or without His6-SIRT5 transfection were treated or untreated by the probe and then subjected to 365 nm UV lamp irradiation for 20 minutes. The mitochondrial parts were extracted prior to SDS-PAGE, fluorescence imaging and Western blotting. As shown in Figure 4B, an intense green fluorescent band appeared in fluorescence image only for the His6-SIRT5 transfected cells treated with the probe. Consistently, the lit up protein could be detected by Western blot using both anti-SIRT5 and anti-His Tag antibodies, confirming the labelled protein is indeed His6-SIRT5. These results demonstrate that the probe, Ni2+-NTA-AF, is applicable to track His6-tagged proteins in different cellular organelles with high selectivity. The His6- Ni2+-NTA system has been explored to image the existing large library of His6-tag proteins in living cells by conjugation of Ni2+-NTA with different fluorophores. Among these probes,

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ACS Sensors the ones we previously reported, Ni-NTA-AC and Ni-NTA-AB27-28, had the advantage of penetrating cell membranes on their own. A green probe we reported here is necessary as it overcomes the drawbacks such as autofluorescence and poor solubility, and is likely to offer great opportunities to image intracellular His6-tagged proteins

Supporting Information. The Supporting Information is available free of charge on the ACS Publication website at DOI: Experimental sections; synthesis and detailed characterization; spectra, ITC, MTT and imaging data (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We acknowledge the financial support from the Research Grants Council of Hong Kong (17333616P and 17307017P), and The University of Hong Kong for an e-SRT on Integrative Biology. We acknowledge Li Ka Shing Faculty of Medicine Faculty Core facility for the support on confocal imaging, and thank the Center for Genomic Sciences (CGS), Li Ka Shing Faculty of Medicine for the mass spectrometry and Typhoon facilities. We are thankful to Prof. Ran-Yi Liu from Sun Yat-sen University Cancer Center for providing the full length XPA plasmid.

ABBREVIATIONS

Figure 4. Fluorescent labelling of His-SIRT5 in vitro and in vivo. (A) HeLa cells and His-SIRT5 transfected HeLa cells were incubated with 25 μM Ni2+-NTA-AF for 30 minutes and subjected to UV laser for 1 minute, the green fluorescence was observed in the mitochondria, where SIRT5 protein was expressed. Scale bar: 10 µm. (B) The corresponding fluorescence SDS-PAGE and Western blot analysis of cell lysate. Lane 1: mitochondrial extraction of HeLa cells; Lane 2: mitochondrial extraction of HeLa cells with Ni2+-NTA-AF; Lane 3: mitochondrial extraction of His-SIRT5 transfected cells; Lane 4: mitochondrial extraction of His-SIRT5 transfected cells with Ni2+-NTA-AF. The green band verified by anti-SIRT5 and anti-His antibodies confirmed the successful labelling of His-SIRT5 inside transfected cells. in living cells.

CONCLUSIONS In summary, we have developed a green fluorescent probe Ni2+NTA-AF by combining a Ni2+-NTA moiety, a fluorescein fluorophore, with an arylazide. The probe selectively binds to His6-tagged proteins, leading to significant fluorescence enhancement upon photoactivation of the arylazide. The incorporation of the arylazide group not only greatly strengthens the binding between the probe and the labelled proteins, but also achieves fluorescence turn-on owing to the structural rearrangement of fluorescein after UV activation. The probe showed good sensitivity and wide linear range on the quantification of His6-tagged proteins. Significantly, the probe could rapidly cross cell membranes to label intracellular His6-tagged proteins at different localizations with high selectivity as demonstrated by XPA and SIRT5 proteins. This green probe Ni2+-NTA-AF, with high quantum yield, less interference from autofluorescence and better solubility, is likely to provide a platform of multicolour tracking for His6-tagged proteins together with the blue Ni-NTA-AC and red Ni-NTA-AB probes which is under investigation in this laboratory.

ASSOCIATED CONTENT

POI, protein of interest; EDT, 1,2-ethanedithiol; CPPs, cell-penetrating peptides; QY, quantum yield; ESI-MS, electrospray ionization mass spectrometry; NMR, nuclear magnetic resonance; MALDI-TOF, matrix assisted laser desorption ionization time of flight mass spectrometry; XPA, Xeroderma pigmentosum group A; ITC, Isothermal titration calorimetry; EDTA, ethylenediaminetetraacetate; PI, propidium iodide; HBSS, Hank’s Balanced Salt Solution.

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