Department of Chemical Sciences, Tata Institute of Fundamental Research, 1 Homi Bhabha Road, Colaba, Mumbai , India.

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1 Samsuzzoha Mondal, 1 Ananya Rakshit, 1 Suranjana Pal, 2 and Ankona Datta*,1 1 Department of Chemical Sciences, Tata Institute of Fundamental Research, 1 Homi Bhabha Road, Colaba, Mumbai , India. 2 Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai , India. * Corresponding Author: Ankona Datta, Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai , India, ankona@tifr.res.in S1

2 SUPPORTING INFORMATION TABLE OF CONTENTS 1. Supplementary figures Page S3 2. Supplementary tables Page S7 3. Supplementary materials and methods Page S7 4. Purity analysis of the DAN-labeled peptides by ESI-LC -MS Page S10 5. References Page S14 S2

3 1. Supplementary Figures Figure S1: Ratiometric response of DAN-probes with 10% PI(4,5)P2-PC mixed vesicles. Top panel: Fluorescence emission spectra of DAN-13aa, DAN-13aa-F163W, and DAN-20aa-F163W (1µM) with increasing concentrations of 10% PI(4,5)P2-PC vesicles ( ex 380 nm). The emission maxima exhibit a blue shift upon PI(4,5)P2 addition in all three cases. Bottom panel: Ratiometric analysis of the fluorescence emission spectra. F 450 /F 520 plotted against concentration of PI(4,5)P2 added. PI(4,5)P2 concentration is calculated as 10% of total phospholipid concentration for the 10% PI(4,5)P2-PC mixed vesicles. The response curve has been fitted to the equation, (F 450 /F 520 ) = (F 450 /F 520 ) max /(1+K d /[PI(4,5)P2]) to obtain K d values. S3

4 Figure S2: Ratiometric response of DAN-peptides toward PI(4,5)P2 in the presence of biologically relevant phospho-anions. (a) DAN-20aa (1 µm) with phospho-anions HPO 2-4, IP3 and ATP (black bars) and DAN-20aa with 10% PI(4,5)P2-PC (35 µm) in the presence of phospho-anions (gray bars). (b) DAN-20aa with 10% PI(4,5)P2-PC (35 µm) followed by addition of IP3 (0-100 µm) shows that IP3 is unable to displace PI(4,5)P2 from the probe. (c) DAN-13aa and DAN-20aa (1 µm) with IP6 (1 mm) (black bars) and DAN-13aa and DAN-20aa with 10% PI(4,5)P2-PC (35 µm) in the presence of IP6 (1 mm) (gray bars). S4

5 Figure S3: Ratiometric response of DAN-13aa and DAN-20aa toward 5% PI(4,5)P2-PC and 5% PI4P-PC vesicles (total phospholipid concentration 80 µm). Figure S4: Ratiometric response of DAN-13aa (1 µm) toward PI(4,5)P2-PC (4:96) and toward PI(4,5)P2-PE-PC (4:21:75) mixed vesicles. S5

6 Figure S5: Response of DAN-13aa (200 nm) toward 5% PI(4,5)P2-PC mixed vesicles in absence and presence of EGFP-PH domain (200 nm). Emission intensity of DAN-13aa at 450 nm has been plotted against PI(4,5)P2 concentration ( ex 380 nm). Figure S6: Comparison between the uptake of the DAN-13aa probe in the HEK293T cells at 37 C and 4 C. Fluorescence signals from the green channels (510 nm 540 nm) have been overlaid on the respective transmission images. Scale bar 10 m. S6

7 2. Supplementary Tables Table S1: Dissociation constants (K d, µm) for the DAN-probes with PI(4,5)P2-PC mixed vesicles containing 5%, 10%, and 20% PI(4,5)P2. 5% PI(4,5)P2-PC 10% PI(4,5)P2-PC 20% PI(4,5)P2-PC DAN-20aa 10±2 4±2 2.6±0.6 DAN-13aa 2.3± ± ±0.2 DAN-20aa-F163W 5±1 4.0± ±0.2 DAN-13aa-F163W 0.7± ± ±0.1 Table S2: Dissociation constants (K d, µm) for DAN-13aa and DAN-20aa with 5% PI(4,5)P2-PC and 5% PI4P-PC mixed vesicles. 5% PI(4,5)P2-PC 5% PI4P-PC DAN-20aa 10±2 21±6 DAN-13aa 2.3±0.4 6±2 3. Supplementary Materials and Methods Unless otherwise mentioned, all chemicals used were of analytical grade, obtained from commercial sources and used without further purification. POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-Lserine), PI(4,5)P2 (L-α-phosphatidylinositol-4,5-bisphosphate, ammonium salt (Brain, Porcine)), PI4P (Lα-phosphatidylinositol-4-phosphate, ammonium salt (Brain, Porcine)), POPS (1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), POPG (1- palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), sodium salt), and PI (L-αphosphatidylinositol, sodium salt) were purchased from Avanti Polar Lipids, Inc. D-myo-Inositol-1,4,5- triphosphate, sodium salt (IP3) was procured from Cayman Chemical, U.S.A. Solid-phase peptide synthesis resin, amino acids, and activating reagents used in peptide synthesis were procured from Novabiochem (Merck Millipore). Acrylodan was obtained from Molecular Probes TM, Thermo Fisher Scientific Inc. Piperidine, N-methylmorpholine, poly(vinyl alcohol), and inositol hexakisphosphate (IP6) were procured from Sigma-Aldrich. Chemicals used in cell culture were procured from Gibco. S7

8 3.1. Peptide synthesis. Peptides were synthesized by using solid state peptide synthesis strategy on Fmoc protected Rink amide resin HL ( mesh, loading 0.74 mmol/g resin) using an automated peptide synthesizer (PS3, Protein technologies Inc., USA). Rink amide resin (100 mg) was de-protected using 20% (v/v) piperidine in dimethylformamide (DMF). For each coupling step, Fmoc protected amino acid (3 eq., mmol), either HBTU (1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) or HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium- 3-oxidhexafluoro phosphate) (3 eq., mmol), and N-methylmorpholine (4% (v/v) in DMF) were used. Both the Fmoc de-protection step followed by the coupling step with the appropriate amino acid were repeated to afford the N-terminal Fmoc protected peptide on the Rink amide resin. After the final Fmoc deprotection, the peptide was cleaved from the resin by treatment with a mixture of trifluoroacetic acid, triisopropylsilane, phenol, water, ethanedithiol, and thioanisole (80 : 2.5 : 5 : 5 : 2.5 : 5) for 5 h. The resins were removed from the mixture and the solution was concentrated under a stream of N 2. The crude peptides were precipitated out in cold methyl-tert-butylether. The peptides were then characterized by MALDI-TOF-MS (Bruker UltrafleXtreme, Bruker Daltonics) Purification and characterization of the DAN probes. DAN labeled peptides were purified on a Varian PrepStar, SD-1 HPLC system, equipped with a Phenomenex Luna C18(2) (250 x 10 mm) semipreparative column. Acetonitrile and water with trifluoroacetic acid (0.1 % v/v) were used as mobile phase and absorption at 220 nm and 380 nm were monitored for product elution. HPLC fractions were characterized by MALDI-TOF-MS to identify fractions containing the desired product. Pure fractions were evaporated under reduced pressure on a vacuum concentrator, (Eppendorf Concentrator Plus, Eppendorf AG, Germany). Purity of the fractions was further confirmed by electro-spray ionization liquid chromatography mass spectroscopy (ESI-LCMS) (LCMS-2020, Shimadzu Corp.) equipped with a Phenomenex Luna C18(2) column (150 x 4.6 mm) using acetonitrile and water with formic acid (0.01 % v/v) as mobile phase. Results of purity analysis obtained by ESI-LC-MS are given from Page S10 onwards Preparation of small unilamellar vesicles (SUVs). Phospholipids were taken in a glass vial in the desired molar ratio and mixed either in chloroform or in a mixture of chloroform:methanol:water (20:9:1). After mixing by vortex, the solvents were evaporated first under N 2 and then overnight under vacuum to obtain lipid films. Lipid films were then hydrated in buffer (20 mm Na-HEPES, 100 mm NaCl, ph 7.4) and taken through 5 freeze-thaw cycles using liquid nitrogen. SUVs were prepared via extrusion by passing the aqueous suspensions through polycarbonate membranes of 100 nm pore diameter (Whatman, GE Healthcare) at 25 C. Formation of vesicles was confirmed by Dynamic Light Scattering (DynaPro, S8

9 Protein Solutions, Wyatt Technology Corp., USA). Concentrations of the phospholipid stock solutions were determined by estimating the total phosphate content using previously reported procedure Preparation of giant unilamellar vesicles (GUVs). GUVs were prepared according to a previously report method. 2 In brief, lipid stock solutions containing phospholipids in desired molar ratio (keeping total phospholipid concentration 1 mg/ml) were prepared either in chloroform or in a mixture of chloroform:methanol:water in 20:9:1. To the lipid solutions, Texas Red DHPE (Molecular Probes TM, Thermo Fisher Scientific Inc.) (0.01 mol%) was added to the lipid solutions to fluorescently label the GUVs. Clean glass coverslips were placed on a hot plate pre-heated to 75 C and aqueous poly(vinyl alcohol) (PVA) solution (5% w/w, 25 µl) was spread on each coverslip. The coverslips were left for drying. This procedure led to the formation of a thin film of PVA on the coverslips. Lipid stock solutions (3 µl) were placed on the PVA films. After the solvent evaporated completely, the films were peeled off and immersed in 150 µl of buffer (20 mm Na-HEPES, 100 mm NaCl, ph 7.4) and left undisturbed for ~ 30 minutes at room temperature. The GUVs formed were released from the PVA film by pipetteaspiration and collected in fresh microtubes Competition experiments with IP3 and other phospho-anions. For the competition experiments, the probes (1 µm) were mixed with either Na 2 HPO 4 (20 mm), IP3 (35 µm), ATP (1 mm), or IP6 (1mM). Fluorescence spectra were recorded for each solution. Then 10% PI(4,5)P2-PC mixed vesicles (total phospholipid concentration 35 µm) were added to each solution and fluorescence spectra were recorded again. To obtain the bar plots (Figure S1a and S1c), F 450 /F 520 values were calculated from the fluorescence spectra and plotted for each phospho-anion. In order to study whether IP3 would be able to displace PI(4,5)P2 from the DAN-peptide, DAN- 20aa (1 µm) was mixed with 10% PI(4,5)P2-PC (total phospholipid concentration 35 µm) and IP3 was added from a stock solution in aliquots up to a final IP3 concentration of 100 µm. Fluorescence spectra were recorded following each IP3 addition. F 450 /F 520 values were plotted against the concentration of IP3 to obtain Figure S1b Cell culture. HEK293T cells were cultured in Dulbecco s Modified Eagle Medium (DMEM, Sigma- Aldrich) supplemented with Fetal Bovine Serum (10%), Penicillin (50 units/ml) and Streptomycin (50 μg/ml) in T25 culture plates at 37 C under humidified air containing 5% CO 2. The cells were plated on home-made glass coverslip bottomed petri plates (35 mm diameter, Tarsons) coated with polylysine (0.1 mg/ml), and incubated at 37 C under 5% CO 2 for 12 h Electroporation of EGFP-PH domain into HEK293T Cells using Neon TM Transfection system. HEK293T cells were washed twice with 1X PBS and detached from the substrate by incubating in 0.25% S9

10 trypsin for two minutes at 37 C. The detached cells were then dissociated by addition of 1 ml of complete DMEM medium (DMEM + 10% FBS) followed by gentle pipetting. The cells were then pelleted by centrifugation at 200 g for 3 minutes. The residual medium was decanted and the cells were re-suspended in 9.5 µl Neon Resuspension Buffer for every 10 million cells. For each electroporation, the cells and 0.5 µl of EGFP-PH domain (from 4.6 µm stock solution) were aliquoted into a sterile microcentrifuge tube. A Neon Tip was inserted into the Neon Pipette and the cell-protein mixture was aspirated into the tip while avoiding air bubbles. The Neon Pipette was then inserted into the Neon Tube containing 3 ml of Neon Electrolytic Buffer E in the Neon Pipette Station. Cells were given four pulses of 10 ms duration at 1400 V. Following this, the cells were plated into home-made glass coverslip bottomed petri plates (35 mm diameter, Tarsons) coated with polylysine (0.1 mg/ml) and incubated at 37 C under 5% CO 2 for 6 hours after which confocal images were recorded Cellular uptake of the DAN-13aa probe at 4 C. HEK293T cells plated on glass coverslips were washed with PBS and incubated at 4 C with DAN-13aa (5 M in cold PBS) for 30 min. Following incubation the cells were first washed with cold PBS and then with room temperature PBS. After the low temperature treatment the cells were imaged immediately at room temperature under a confocal microscope. In a control experiment, cells were incubated at 37 C with same concentration of DAN-13aa for 30 min followed by washing with PBS at room temperature before imaging. 4. Characterization and purity analysis of DAN probes Solvent: Solvent A: water with 0.01% formic acid; solvent B: acetonitrile with 0.01% formic acid. Gradient: 0-5 min: 5% solvent B; 5-45 min: % solvent B; 50 min: 100% solvent B; 55 min: 10% solvent B; 60 min: 10% solvent B. Column: Phenomenex Luna C18(2) a) DAN-13aa: C(-DAN)VVQRLFQVKGRR Molecular weight: (Calc. for C 84 H 137 N 27 O 16 S) MALDI-TOF-MS, m/z: (M+H) + S10

11 LC elution trace at 380 nm ESI-MS (+ve mode) of peak (25.5 min) in LC b) DAN-20aa: KHVVPNEVVC(-DAN)QRLFQVKGRR Molecular weight: Da (Calc. for C 120 H 194 N 38 O 26 S). MALDI-TOF-MS, m/z: (M+H) + S11

12 LC elution trace at 380 nm ESI-MS (+ve mode) of peak (26 min) in LC c) DAN-13aa-F163W: C(-DAN)VVQRLWQVKGRR Molecular weight: Da (Calc. for C 86 H 138 N 28 O 16 S). MALDI-TOF-MS, m/z: (M+H) + S12

13 LC elution trace at 380 nm ESI-MS (+ve mode) of peak (26.5 min) in LC d) DAN-20aa-F163W: KHVVPNEVVC(-DAN)QRLWQVKGRR Molecular weight: Da (Calc. for C 122 H 195 N 39 O 26 S). MALDI-TOF-MS, m/z: (M+H) + S13

14 LC elution trace at 380 nm ESI-MS (+ve mode) of peak (25.5 min) in LC 5. References 1) Gupta, S.; Mondal, S.; Mhamane, A.; and Datta, A. Smart lanthano proteins for phospholipid sensing. Inorg. Chem. 2013, 52, ) Weinberger, A.; Tsai, F.C.; Koenderink, G.H.; Schmidt, T.F.; Itri, R., Meier, W.; Schmatko, T.; Schröder, A; and Marques, C. Gel-assisted formation of giant unilamellar vesicles. Biophys. J. 2013, 105(1), pp S14