Chemicals and reagents
1,2-dioleoyl-sn-glycero-3-phosphocholine (PC), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (PG), 1,2-dipalmitoyl-sn-glycero-3-phospho-ethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) and N-(lissamine rhodamine B sulfonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (rhodamine-PE) were obtained from Avanti Polar Lipids. Nitrilotriacetic acid (Ni2+-NTA)-chelating Sepharose and Superose 6 increase 10/300 GL were purchased from GE Healthcare. 1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate) (Dil), N,N′-Dimethyl-N-(Iodoacetyl)-N′-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Ethylenediamine (IANBD amide) and Oregon GreenTM 488 (OG) maleimide were obtained from ThermoFisher. All other chemicals were acquired from Sigma.
Plasmids
pET28a-MSP1D1 was a gift from Dr. Steven Sligar7. pET28a-NW30 and pET28a-NW50 were gifts from Dr. Gerhard Wagner11. All other constructs in this work were made using the In-Fusion® HD Cloning Kit (Takara Bio USA). SpyCatcher002 was synthesized as a gBlocks gene fragment (IDT), and inserted together with SpyTag002 into pET28a-MSP1D1, yielding pET28a-spMSP1D1. pET28a-spNW30 and pET28a-spNW50 were made by replacing MSP1D1 in pET28a-spMSP1D1 with NW30 and NW50 amplified from pET28a-NW30 and pET28a-NW50. pET28a-spNW80 and pET28a-spNW100 were generated by inserting NW30 and NW50 into pET28a-spNW50. pET28a-spNW15 and pET28a-spNW25 were built by truncation from pET28a-spNW30. spMSP protein sequences are described in Supplementary Table I. All the spMSP plasmids have been deposited in Addgene: spMSP1D1 (ID: 173482), spNW15 (ID: 173483), spNW25 (ID: 173484), spNW30 (ID: 173485), spNW50 (ID: 173486), spNW80 (ID: 173487), spNW100 (ID: 173488).
Full-length cpx2 and the fragment encoding the cytoplasmic domain of syt1 were cloned into pGEX-4T43. Full-length EIIAGlc was cloned into pBad33 with a C-terminal His-tag33. For site-directed labeling, native cysteines were substituted with alanine. Then, single cysteines were introduced at Phe234 of syt1, Lys134 of cpx2, Thr96 of EIIAGlc.
Proteins
syt1, cpx2, EIIAGlc, MSP1D1, and SNAREs were expressed in BL21 STARTM (DE3) and purified using GSTrap and Ni2+-NTA columns20,25,44,45,46,47. To produce spMSP1D1,spNW15, spNW25, spNW30, spNW50, spNW80, and spNW100, plasmids were transformed into BL21 STARTM (DE3) cells that were grown in LB supplemented with Km (50 mg/ml) to OD600 ~ 0.7. Protein expression was induced with 0.2 mM IPTG at 16 °C overnight. Bacteria were harvested by centrifugation at 3450 × g for 20 min, resuspended in buffer A (50 mM Tris-HCl (pH 8),100 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol), and lysed on ice using a Branson cell disrupter (60% duty cycle, 45 secs). Cell lysates were clarified by centrifugation at 12,000 × g for 45 mins. The supernatants were loaded onto a 1 ml Ni2+-NTA column (GE Healthcare), followed by extensive wash (20 column volume) using buffer B (50 mM Tris-HCl (pH 8), 20 mM Imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol). Proteins were eluted in buffer C (50 mM Tris-HCl (pH 8), 500 mM Imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol), desalted in buffer A using PD MiDiTrap G-25 (GE Healthcare), and stored at −80 °C.
Fluorescent labeling of proteins
Purified syt1, cpx2, and EIIAGlc were desalted using Zeba Spin columns (Thermo Fisher) in buffer D (50 mM Tris-HCl, pH 8, 100 mM NaCl, 5% glycerol) and labeled with a 3-fold excess of IANBD amide or OG maleimide in the presence of TCEP (0.2 mM) at room temp for 2 h. Free dyes were removed by passing through Zeba Spin columns in buffer A.
For fluorescence fluctuation spectroscopy experiments, spMSP1D1, spNW30, and spNW50 were desalted using Zeba Spin columns in PBS buffer and labeled with a 3-fold excess of fluorescein isothiocyanate (FITC) at room temp for 2 h. Free dyes were removed by passing through Zeba spin columns in buffer A.
Nanodiscs
A step-by-step protocol describing nanodisc reconstitution can be found at Protocol Exchange48. To optimize the condition for cND reconstitution, spMSPs were incubated with PC lipids at the indicated ratio in buffer A containing 0.05% DDM. To incorporate v-SNARE into nanodiscs, v-SNARE, MSPs, and PC lipids, were mixed at a ratio of 2:5:60 for spMSP1D1, 2:5:600 for spNW25, and 2:5:2000 for spNW50 in buffer A containing 0.05% DDM. For lipid mixing assays, v-SNARE nanodiscs were reconstituted with 12% PE, 40% PS, 45% PC, 1.5% NBD-PE, and 1.5% rhodamine-PE. To monitor the binding of syt1 to lipids via fluorescence spectrometry, nanodiscs were made using 70% PC and 30% PS. To study the interaction of cpx2 and EIIAGlc via FRET, nanodiscs were made using 1% Dil, 69% PC, and 30% PS or PG. Samples were kept on ice for 30 min, and detergents were slowly removed with BioBeads (1/3 volume) and gentle shaking (4 °C, overnight). cNDs were purified by SEC in Superose 6 10/300 in buffer A and stored at −80 °C.
Crosslinking experiments
cNDs (0.5 µM) were desalted in PBS using Zeba Spin columns and incubated with the indicated concentration of ethylene glycol bis(succinimidyl succinate) (EGS, ThermoFisher) at room temperature for 1 h. Reactions were quenched by the addition of 100 mM Tris-HCl (pH 8), and samples were subject to SDS-PAGE analysis. Image Lab software (BioRad) was used to collect data and Image StudioTM Lite Ver 5.2 (LI-COR) to analyze data.
Negative stain electron microscopy
NDs (10 µg/ml) were applied onto Formvar/carbon-coated copper grids (01754-F, Ted Pella, Inc.) that were glow discharged (15 mA, 25 s) using PELCO easiGlowTM (Ted Pella, Inc). After 30 s, samples were stained with 0.75% uranyl formate for 1 min. Grids were imaged on a ThermoFisher Science Tecnai G2 TEM (100 kV) equipped with a Veleta CCD camera (Olympus). TIA (FEI) software was used to collect data and ImageJ2 Fiji to analyze data.
Mass spectrometry
Protein samples were incubated with trypsin at 37 °C overnight. The resulting peptide samples were injected for inline pepsin digestion and the resulting peptides were identified using tandem MS (MS/MS) with an Orbitrap mass spectrometer (Q Exactive, ThermoFisher). Following digestion, peptides were desalted on a C8 trap column and separated on a 1 h linear gradient of 5–40% B (A is 0.3% formic acid and B is 0.3% formic acid 95% CH3CN). Product ion spectra were acquired in a data-dependent mode such that the 10 most abundant ions were selected for the product ion analysis by higher-energy collisional dissociation between survey scans. Following MS2 acquisition, the precursor ion was excluded for 16 seconds. The resulting MS/MS data files were submitted to Proteome Discoverer (Thermo, version 2.5.0.400) for database searches and peptide identification. The precursor and fragment mass tolerances were set to 10 ppm and the false discovery rate was set to 5%. The MS/MS spectra identifying peptides were verified by manual inspection. The Proteome Discoverer results were used as input for skyline (version 20.2.0.343) to quantify peak areas.
Fusion assays
For the cargo release assay, t-SNARE vesicles encapsulated with glutamate or fluorescence dextran were prepared as described previously20,25. The reaction was initiated by incubation of v-SNARE nanodiscs (0.5 µM) and t-SNARE vesicles (1 µM) in reconstitution buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl) at 37 °C for 30 mins. For the glutamate release assay, the glutamate sensor iGluSnfR49,50 (0.1 µM) was added to the reaction mixture. For the dextran release assay, samples were centrifuged at 50,000 × g for 30 min, and the supernatants were carefully collected for further analysis. The fluorescence of iGluSnFR and dextran was quantified using a Synergy H1M plate reader with excitation at 460 nm and emission at 530 nm. The percentages of cargo release were determined by normalization of data to the maximal release after the addition of 0.5% DDM to each sample.
For the lipid mixing assay, v-SNARE vesicles were prepared by detergent removal20,25. Briefly, purified synaptobrevin-2 (syb-2) were incubated with lipids (12% PE, 40% PS, 45% PC, 1.5% NBD-PE and 1.5% rhodamine-PE) in the reconstitution buffer supplemented with 0.1% DDM. Detergents were slowly removed by addition of Biobeads (1/3 volume) and gentle shaking (4 °C, overnight). Liposomes harboring v-SNAREs were extruded through an Avanti extruder with 200 nm filter and further purified using PD MiDiTrap G-25 (GE Healthcare) in reconstitution buffer. The lipid mixing assays were performed by incubation of v-SNARE vesicles or nanodiscs bearing the FRET reporter (NBE-PE and Rhodamine-PE) with t-SNARE vesicles in the presence of C2AB (1 μM) and Ca2+ (0.5 mM). The NBD signal was monitored for an additional 1 h using a Synergy H1M plate reader with excitation at 460 nm and emission at 530 nm. The efficiencies of membrane fusion were determined by normalization of data to the maximal release after the addition of 0.5% DDM to each sample. BioTek Gen5 software was used to collect data and Graphad 8 to analyze data.
Fluorescence spectroscopy
NBD- and OG-labeled proteins (10 nM) were incubated with nanodiscs at the indicated concentrations in reconstitution buffer. The fluorescence spectrum of samples was collected on a Synergy H1M plate reader with excitation at 460 nm and emission from 500–650 nm.
Fluorescence fluctuation spectroscopy
spMSPs were labeled with FITC and then used for cND reconstitutions. The molecular brightness of FITC-labeled spMSPs and cNDs (100 pM) in reconstitution buffer were quantified as counts per second per molecule (cpsm) via photon counting histogram using an ISS Alba v5 laser scanning microscope (ISS, Inc.). Samples were excited using a pulsed 488 nm laser, and photon counts were detected with an avalanche photodiode (APD) (Excelitas, SPCM-AQRH-15). Data were analyzed using the ISS vista vision software.
Other methods
SDS-PAGE and Native PAGE electrophoresis were performed using 4–15% TGX stain-freeTM protein gels (Bio-Rad) and imaged using the Image Lab software on a GelDoc Go system (Biorad). Size-exclusion chromatography (SEC) was carried out on an AKTA pure 25 L using Superose 6 10/300 (GE Healthcare) in 50 mM Tris-HCl, pH 8, 100 mM NaCl, 5% Glycerol45,46. Unicorn 7 software was used to collect data and Graphad 8 to analyze data. Density gradient flotation was performed on a Beckman Optima XE-90 Ultra using an Accudenz step gradient (Accurate Chemical & Scientific Corporation)20,25.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
