Site-directed mutagenesis and protein labeling

Site-directed mutagenesis was performed using the QuikChange protocol (Agilent) and confirmed by sequencing. SecY_MKEG, SecY_MK,R357EEG, SecA, SecB and full length proOmpA were produced as described previously (Deville et al., 2011 (link); Gold et al., 2007 (link); Whitehouse et al., 2012 (link)). Different proOmpA lengths were produced adopting existing methods (De Keyzer et al., 2002 (link)). OmpA lacking the SS was purified as described in Schiffrin et al. (2016 (link)). SecY_MKEG was produced in the same way as wild-type, then labelled for 45 mins on ice at 50 µM with 100 µM each of Alexa 488-C₅-maleimide and Alexa 594-C₅-maleimide (Invitrogen). The reactions were quenched with 10 mM DTT, and excess dye removed by gel filtration (Superdex-200, GE Healthcare, UK). Labelling efficiencies were between 75% and 90% for each dye, as determined using the manufacturer's quantification method and assuming a molar extinction coefficient of 70,820 cm⁻¹ for SecY_MKEG.