MscL Cysteine Mutant Generation and Purification

Cysteine mutants were generated for residues 14–43 and 72–107 in MscL, covering the predicted length of TM1 and TM2, respectively. Mutagenesis was performed by oligonucleotide mismatch site-directed mutagenesis using the Transformer kit (CLONTECH Laboratories, Inc.) and confirmed by dideoxy DNA sequencing. Mutant channels were expressed and purified as follows: the construct MscL-pQE32 containing MscL with the RGS-(4× His) epitope at the NH₂ terminus was used to transform E. coli XL-1 blue cells (Stratagene) using standard chemical methods. After protein expression was induced by addition of 1 mM IPTG, membranes were solubilized in PBS containing dodecyl maltoside (DDM) at room temperature, spun-down at 100,000 g for 1 h and purified with a Co²+-based metal-chelate chromatography resin (Talon resin; CLONTECH Laboratories, Inc.). Unless specifically noted, the purified mutant protein was spin labeled overnight with methanethiosulfonate spin label (Toronto Research) at a 10:1 label/channel molar ratio and reconstituted at a 500:1 lipid/channel molar ratio by dilution in PBS (Cuello et al. 1998).