Protein handling

2.2.1 Basic methods Bacterial transformation

Escherichia coli BL21 DE3 cells (electrocompetent from Stratagene/Agilent Tech-nologies) were transformed with plasmids carrying constructs listed in Table 2.1 us-ing a modified protocol from Dower et al. (1988). All used protein constructs were cloned into the NdeI/XhoI restriction sites of the pET28a expression vector (Novagen, 2003). For electroformation, bacteria (∼ 70 µl) were thawed on ice and then mixed with 50–100 ng of plasmid DNA in an electroporation cuvette (0.2 cm gap, BioRad).

The electroporation was performed with MicroPulser (BioRad) with an electrical pulse 2.5 kV. Then, bacteria were mixed with 1 ml of preheated LB medium (Bertani, 1951;

without glucose) and incubated at 37 °C shaking for ∼ 45 min. Afterwards, bacteria were plated on a LB agar plate (1.8 % w/v agar dissolved in the LB medium) with kanamycin, and grown overnight at 37 °C.

Table 2.1. Protein constructs. All protein sequences were derived from Rattus Norvegicus unless stated otherwise. Constructs were cloned into a pET28a expression vector.

syb 1–96 1–96 S28C Margittai et al.

WT syb 1–116 - Pabst

Δ84 syb 1–116 Δ84 Siddiqui et al.

AA syb^a 1–116 I45A, M46A Wiederhold et al.

- 1–206 C84S, C85S,

C90S, C92S Fasshauer et al.

SNAP-25 C84S, C85S,

- 1–206 C90S, C92S, Margittai et al.

S130C

syntaxin Syntaxin-1A 183–288 - Schuette et al.

NSF^a,b

SNAP alpha 1–295 - Winter et al.

aexpressed and purified by Ursel Reis or Dr. Halenur Yavuz (Yavuz, 2015)

bChinese hamster sequence (Cricetulus griseus)

cbovine sequence (Bos taurus)

Protein expression

The expression and purification of proteins was done essentially as described in Her-nandez (2012) and Yavuz (2015, especially syntaxin purification protocol). For pro-tein expression a colony was picked from a LB agar plate and grown overnight in LB medium at 37 °C. On the next day, this pre-culture was used for a large scale expression in TB medium (initially presented by Tartof and Hobbs, 1987 with further modifica-tions reported in Cold Spring Harbor Protocols, 2006; supplemented with 50 µg/ml kanamycin). Cells were grown shaking at 37 °C until they reached OD600 0.8–1.0, and

then expression was induced with 0.5 mM IPTG. Recombinant protein was expressed for 4 h at 37 °C and then harvested via centrifugation (20 min, 4 °C, 4,100×g), resus-pended in resuspension buffer (Table A.6,∼100 ml), and stored at −20 °C.

Protein purification

For protein extraction and purification bacteria were thawed and subsequently the ex-traction buffer (Table A.6, 100 ml) supplemented with lysozyme (4 mg/l of initial cul-ture), MgCl2 (1 mM), DNaseI (tip of a spatula), protease inhibitor cocktail tablet (cOmplete, EDTA-free, Roche) was added. Afterwards, this mixture was incubated for 15 min at room temperature. Cells were then lysed with a sonicator (Branson Sonifier 450, microtip limit, 50 % duty cycle, 4×40 strokes), supplemented with 6 M urea, and incubated for around 15 min. Protein was later harvested by centrifugation (60 min, 4 °C, 25,000×g) and then incubated for at least 2 h with Ni-NTA agarose beads at 4 °C (Qiagen, 12 ml beads per 6 l culture). Beads with bound His-tagged protein were collected on Econo-Column (BioRad), washed with the wash buffer and protein was eluted with the elution buffer containing concentrated imidazole (both so-lutions in Table A.6). After overnight dialysis (dialysis buffer, Table A.6, supplemented with thrombin for the His-tag cleavage 5 mg/ml in 50 % glycerol, 1 U/µl, 100 ml for 5 ml of protein solution; MWCO 8 kDa), protein was further purified by ion exchange chro-matography (with the ÄKTA system) using a NaCl gradient (from 50 mM to 500 mM) for elution (buffer with HEPES 20 mM, pH 7.4, DTT 1 mM, EDTA 1 mM, and supple-mented with OG for syb 1–116 and CHAPS for syntaxin). Depending on the protein isoelectric point (determined in ExPASy database, Gasteiger et al., 2005), anion ex-changer (for syntaxin and SNAP-25) or cation exex-changer (for synaptobrevin) was used.

Protein purity and His-tag cleavage efficiency was evaluated by Tricine–SDS-PAGE fol-lowed by Coomassie blue staining (see sectionTricine–SDS-PAGE and Coomassie blue staining). Fraction concentration was calculated based on the absorbance at 280 nm (NanoDrop 1000 spectrophotometer, Thermo Scientific), and extinction coefficients and molecular weight determined by ProtParam tool available in the ExPASy database (Gasteiger et al., 2005) with the Beer–Lambert law (Equation 1). Aliquoted protein was then snap frozen in liquid nitrogen and stored at−80 °C until use.

Tricine–SDS-PAGE and Coomassie blue staining

Proteins were analysed by electrophoresis with Tricine–SDS-PAGE according to the pro-tocol by Schägger and von Jagow (1987) with following modifications. Stacking and separating gel contained 4 % and 10 % acrylamide, respectively, and the electrophoretic run was performed at 60 V for 15 min followed by 120 V for∼45 min. For visualisation the polyacrylamide gel was stained with a Coomassie blue by briefly boiling the gel in the Coomassie solution in the microwave and subsequent ∼ 5 min incubation in the room temperature. Next, the gel was destained in 2 steps by gently shaking for 5 min in the destaining solution 1 and for several hours in the destaining solution 2 (Table A.6).

2.2.2 Protein labelling

Fluorescent dyes (Table A.1) were coupled to single cysteine mutants of syb 1–96 and SNAP-25 according to the manufacturers instructions (Molecular Probes, 2006). For the Oregon Green 488 (OG488) iodoacetamide, methanol was used as a solvent, and for the Texas Red (TR) maleimide, DMSO was utilised. Dyes were added in 6–10× molar excess to the protein solution (with DTT dialysed out) and incubated for 2 h at room temperature. The unreacted dye species were then removed by size exclusion on PD-10 columns. Dye concentration (c) was calculated with the Beer–Lambert law (Equation 1), by measuring dye absorbance (A) at the absorption maximum wavelength on NanoDrop 1000, and with light path length (l) and dye extinction coefficient (ε).

A=εcl ⇒ c= A

εl (1)

Protein concentration was determined with a Pierce 660 nm Protein Determination Kit according to manufacturers instructions (Pierce Biotechnology, 2013), with absorbance readout on a microplate reader (Genios Pro, TECAN). Degree of labelling (DOL, Equa-tion 2) was obtained by comparison of dye (c_dye) and protein (c_protein) concentration in the final fractions.

DOL = cprotein

c_dye (2)

2.2.3 SNARE acceptor complex formation

SNARE acceptor complex (the so calledΔN complex) consisting of SNAP-25, syntaxin lacking its N-terminal domain, and syb 49–96 was assembled from purified monomers as described in Pobbati et al. (2006) with the use of OG as in Hernandez et al. (2012).

Briefly, monomers were mixed in the molar ratio 1:1:1.5, respectively, and the OG con-centration was adjusted to 1 % (w/v). After overnight incubation, complex was purified by ion exchange chromatography with anion exchanger in the presence of 1 % (w/v) OG with a 2-step linear NaCl gradient (complex eluted at∼300–400 mM salt). Purity of the complex was assessed by analysis of unboiled protein sample with Tricine–SDS-PAGE and Coomassie blue staining, and activity was checked by determination of syb 1–96 (labelled with OG488) binding efficiency with fluorescence anisotropy measure-ment (as described in Section 2.4). The purified complex was then snap frozen and stored at−80 °C until use. Labelled complex was essentially formed in the same way, with the exception of SNAP-25 being replaced with a S130C mutant coupled to Texas Red.