Imidazole

All recombinant proteins were expressed in Escherichia coli BL21 (DE3) grown in LB medium at 37 °C to an A₆₀₀ of 0.7. The protein expression was induced with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Cells were then grown for another 5 hours at 25 °C for soluble proteins, SNAP-25 and apoA1 or 16 hours at 16 °C for proteins with the transmembrane domain, syntaxin-1a and VAMP-2, respectively. Pellets were resuspended in phosphate buffered saline (PBS) or PBS with 0.3% triton (PBST) with the protease inhibitor, 4-(2-Ainoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and additional dithiothreitol (DTT) for single cysteine mutants of syntaxin-1a or VAMP-2. Cells were lysed by sonication, after which lysates were centrifuged at 13,000 rpm for 30 min at 4 °C to remove insoluble fractions. Supernatants containing glutathione S-transferase (GST)-fused recombinant proteins, syntaxin-1a, SNAP-25, and VAMP-2 were then incubated with glutathione sepharose beads (GE healthcare) while 6× His-tagged SNAP-25 and apoA1 were incubated with Ni-NTA agarose beads (Qiagen) for 1 hour at 4 °C. After washing with PBS or PBST, GST-fusion proteins were eluted with thrombin (0.02 unit/µl, Sigma) in PBS or PBS with 0.8% octyl β-D-glucopyranoside (PBS-OG) for membrane proteins and 6× His-tagged proteins were eluted with 200 mM imidazole (Sigma) in PBS. The elution buffer for single cysteine mutant proteins contained 2 mM DTT. For pull down of the trans-SNAREpin sandwiched between two nanodiscs, His-tag-free apoA1 was obtained with thrombin cleavage of His-tag (Figure S1). For the smFRET study of the trans-SNAREpin, single cysteine mutants of syntaxin-1a (I203C or V241C) and VAMP-2 (Q33C or A72C) were desalted with PD 10 column (GE healthcare) to eliminate free DTT in solution, then incubated with 10× molar excess of maleimide-derivative fluorophore, Cy5 or Cy3, respectively for overnight at 4 °C. Unreacted free fluorescence labels were removed by the PD 10 column and the labeling efficiency of each SNARE protein was measured with spectrophotometry (Beckman). Extinction coefficients of Cy5 (250 000 M⁻¹ cm⁻¹ at 650 nm) and Cy3 (150 000 M⁻¹ cm⁻¹ at 552 nm) were used to calculate the concentration of the fluorophore. The detergent compatible Lowry assay (DC assay, Bio Rad) was recruited to determine the concentration of SNAREs. The labeling efficiency for each protein was 46% and 40% for syntaxin-1a I203C and V241C and 55% and 62% for VAMP2-2 Q33C and A72C, respectively. For spin labeling with MTSSL (Enzo Life Sciences) of VAMP-2 cysteine mutants, 10× molar excess of MTSSL was added to GST-VAMP-2 cysteine mutants on glutathione sepharose beads right after rapid washing with DTT free PBST before overnight incubation at 4 °C. Free MTSSL was washed out before eluting spin-labeled VAMP-2 with thrombin.