Expression of VDAC-2
The cDNA for human voltage-dependent anion channel isoform 2 (VDAC-2) with the GenBank accession number BC000165 was obtained as a full-length clone from the Mammalian Gene Collection (OpenBioSystems, Huntsville, AL). VDAC-2 with a C-terminal His-tag (VDAC-2(1–294)-Leu-Glu-His-His-His-His-His-His) was cloned by standard PCR methods into the NheI/XhoI sites of pET21d (Novagen). Site-directed mutagenesis was carried out to change the codons for the second and the third residues (Ala-Thr) of VDAC-2 to be GCTACT to have optimized protein expression (Qiagen). VDAC-2 was expressed in Rosetta 2(DE3) cells by induction with 1mM IPTG (Gold Biotechnology) at 37 °C for 12 hours.
Purification of VDAC-2
Cells were resuspended in lysis buffer (50 mM Tris-HCl, 250 mM NaCl, 5 mM 2-mercaptoethanol (Sigma), protease inhibitors tablets (Roche), pH 6.8) and lysed by sonication. The VDAC-2 containing inclusion bodies were isolated using sucrose cushion centrifugation (50 mM Tris-HCl, 500 mM NaCl, 50 % sucrose, pH 6.8). The inclusion bodies were dissolved in denaturing buffer (8 M urea, 50 mM Tris-HCl, 100 mM NaCl, 20 mM imidazole, pH 7.5), applied to Ni-agarose resin (Qiagen) and VDAC-2 was eluted with elution buffer (8 M urea, 50 mM Tris-HCl, 100 mM NaCl, 250 mM imidazole, pH 7.0). Purified VDAC-2 in elution buffer was precipitated by dialysis against 4 liters of dialysis buffer (50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 5 mM DTT, pH 7.0) in a 6,000–8,000 molecular weight cutoff (MWCO) dialysis membrane. Precipitated VDAC-2 was isolated by centrifugation at 12,000 g.
Refolding and Purification of VDAC-2 in LDAO detergent
Purified precipitated VDAC-2 was dissolved in guanidine hydrochloride buffer (100 mM NaPi, 100 mM NaCl, 6 M GuHCl, 5 mM TCEP, 1 mM EDTA, pH 6.7) at a concentration of 3 mg/ml. VDAC-2 was refolded at 4 °C by dropwise dilution of one volume VDAC-2 solution into 10 volumes refolding buffer (25 mM NaPi, pH 6.8, 100 mM NaCl, 1 mM EDTA, 5 mM TCEP, 1.5% (64.5 mM) LDAO (Anatrace and FBReagents)) with stirring. The final ratio of VDAC-2 protein to LDAO micelles is about 1:90 assuming the aggregation number of LDAO micelle is 76. After overnight stirring at 4 °C the refolded VDAC-2 sample was dialyzed against 4 liters of buffer (25 mM NaPi, 1 mM EDTA, 5 mM DTT, pH 6.8) for 12 to 16 hours. Little amount of precipitated VDAC-2 was removed using centrifugation at 12,000 g, followed by filtration with a 0.22-μm membrane.
Cation exchange chromatography was employed to isolate properly folded VDAC-2 protein in LDAO detergent micelles. The sample was loaded onto a 30 ml SP sepharose HP column equilibrated with buffer A (25 mM NaPi, 1 mM EDTA, 5 mM DTT, 0.1% LDAO, pH 7.0). VDAC-2 was eluted during a 5%–55% gradient with buffer B (25 mM NaPi, 1 mM EDTA, 5 mM DTT, 0.1% LDAO, 1M NaCl, pH 6.2) at about 35% buffer B. Pure VDAC-2 fractions were pooled and concentrated to the desired concentration using 30k molecular weight cutoff (MWCO) concentrators.
VDAC-2 was further purified by gel filtration chromatography using a superdex 200 prep grade column (GE Healthcare). The column was equilibrated in NMR buffer (25 mM NaPi, 100 mM NaCl, 20 mM TCEP, 0.1% LDAO, EDTA 1 mM, pH 6.8). After final purification, the sample was concentrated using 10 k MWCO concentrator. The LDAO detergent concentration was monitored by 1D 1H-NMR spectroscopy and adjusted by a series of dilution and concentration steps with buffer A. The final sample conditions were 25 mM NaPi (pH 6.8), 100 mM NaCl, 20 mM TCEP, ~250 mM LDAO and 0.5–0.8 mM VDAC-2.
Incorporation of VDAC-2 in DMPC nanodiscs
Following the protocol for preparing VDAC-1 in DMPC nanodiscs [27
], VDAC-2 was first refolded in LDAO detergent micelles and subsequently assembled into DMPC nanodiscs. The plasmid coding for the N-terminal deletion mutant (MSP1D1) of the membrane scaffold protein (MSP1) was obtained from the non-profit plasmid repository Addgene deposited by Stephen Sligar [28
]. Expression and purification of MSP1D1 was essentially done as described in the reference [28
]. For the incorporation of VDAC-2 into nanodiscs, MSP1D1 protein, refolded VDAC-2 in LDAO detergent micelles and sodium cholate solubilized DMPC lipids were incubated for 1 hr at room temperature prior to the removal of the detergents by the addition of biobeads SM2 (BioRad). The ratio of lipid to MSP1D1 was optimized by analytical size exclusion chromatography to account for displacement of lipid molecules by VDAC-2. A 1:8:640 ratio for VDAC-2/MSP1D1/DMPC lipid was found to yield the highest fraction of assembled nanodiscs. Reconstituted VDAC-2 embedded in nanodiscs was isolated by Ni-NTA affinity chromatograph followed by size-exclusion chromatography onto a Superdex 200 prep grade gelfiltration column (GE Healthcare) equilibrated in 25 mM NaPi
(pH 6.8), 100 mM NaCl, 0.5 mM EDTA. The final concentration of [U
N]VDAC-2 and [U
N]VDAC-2 were estimated to be ~200 and 500 μM, respectively.
All NMR experiments were performed on Bruker spectrometer operating at field strengths of 750, 800 and 900 MHz. All spectrometers were equipped with cryogenically cooled probes. For VDAC-2 solubilized in LDAO detergent micelles, 2D [15
H]-TROSY, 3D TROSY-HNCA, 3D [1
N-TROSY and [15
H]-TRACT experiments [29
] were recorded at 30 °C. For VDAC-2 embedded in DMPC nanodiscs, 2D [15
H]-TROSY, 3D [1
N-TROSY and [15
H]-TRACT experiments were acquired at 30 °C and 40 °C.