To obtain weak crown mutants, we designed a mutation in the crown structure of the A3B3 ring. On the basis of the 3D structure of A3B3 and V1 in E. hirae (18), we selected the amino acid for mutation at the contact interfaces of three A1B1 units in the β-barrel region (B-L65Y, which is not conserved in F-, A-, and V-ATPases). Mutagenesis of the L65Y of B subunit was performed using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) (20).

An E. coli cell-free protein expression system was used to synthesize the A, B [or B(L65Y)], and DF complexes [D(T60C/L67C)F mutant for single-molecule analysis (20)] using mixtures of respective plasmids harboring the corresponding genes with a modified natural poly-histidine (MKDHLIHNHHKHEHAHAEH) affinity tag, tobacco etch virus cleavage site (EHLYFQG), and linker (SSGSSG) sequences at the N terminus (17, 18). The cell-free lysate reaction mixture was loaded onto a HisTrap HP column (GE Healthcare, Little Chalfont, UK) equilibrated with buffer A [20 mM tris-HCl, 1 M NaCl, and 20 mM imidazole (pH 8.5)], and bound proteins were eluted with buffer B [20 mM tris-HCl, 500 mM NaCl, and 500 mM imidazole (pH 8.0)]. To obtain nontagged samples, the proteins were treated with tobacco etch virus protease at 4°C. The reaction solution was dialyzed overnight against buffer C [20 mM tris-HCl, 500 mM NaCl, 20 mM imidazole, and 2 mM dithiothreitol (DTT) (pH 8.5)]. The dialysate was loaded onto a second HisTrap HP column equilibrated with buffer A, and the flow-through fractions containing the nontagged proteins were pooled. The sample buffer was exchanged with buffer D [20 mM tris-HCl, 20 mM NaCl, and 2 mM DTT (pH 8.5)] using a HiPrep 26/10 desalting column (GE Healthcare), loaded onto a HiTrap Q HP column (GE Healthcare) equilibrated with buffer D, and eluted with a linear gradient of 20 to 1000 mM NaCl.

For wild-type A3B3 and mutant A3B(L65Y)3 complexes. Eluted samples were further purified using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare) equilibrated with buffer E [20 mM tris-HCl, 10% glycerol, 100 mM NaCl, and 2 mM DTT (pH 7.5)]. Purified samples were concentrated with an Amicon Ultra-10 filter with a molecular weight cutoff of 10,000.

For the DF complex. The eluted sample was further purified using a HiLoad 16/60 Superdex 75 pg column (GE Healthcare) equilibrated with buffer F [20 mM tris-HCl, 150 mM NaCl, and 2 mM DTT (pH 8.0)]. Purified sample was concentrated with an Amicon Ultra-30 filter with a molecular weight cutoff of 3000.

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