Intramolecular disulfide formation in LmrP<i>-dc</i> (I34C & V240C) and mutants derived thereof.

<p><b>A</b>, Ethidium efflux in intact cells expressing Wt LmrP, LmrP<i>-cl</i> or LmrP<i>-dc</i> show that, similar to Wt LmrP, the mutants are transport active. The measurements were performed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038715#pone-0038715-g003" target="_blank">Fig. 3A</a>. The traces represent results of three independent experiments (n = 3). <b>B,</b> Cysteine crosslinking between I34C and V240C in LmrP<i>-dc.</i> RSOVs containing LmrP<i>-dc</i> (2 µg protein/µl) in 100 mM KP_i buffer, pH 7.0, plus 5 mM MgSO₄ were exposed to 0.5 mM copper phenanthroline (<i>Cu/Ph</i>) in the presence or absence of 97.9 µM benzalkonium chloride (<i>BC</i>) for 5 min at 10°C. The reactions were stopped by the addition of excess (10 mM) of the thiol alkylator N-ethylmaleimide followed by incubation for 2 min at 10°C. Total protein (5 µg) of RSOVs from the crosslinking reactions were mixed with 5 × SDS sample-loading buffer devoid of dithiothreitol (DTT) (<i>lane 1–4</i>) or containing 2 mM DTT (<i>lane 5</i>), and separated on 11% SDS-PAGE. The SDS-PAGE gel was subsequently subjected to Western blot transfer onto a HybondP membrane. Transferred proteins were probed with anti-His₅ antibody. Upper band: uncrosslinked protein; Lower band: crosslinked protein. <b>C,</b> Time course for disulfide formation between I34C and V240C. The crosslinking in RSOVs containing LmrP<i>-dc</i> was followed for 10 min at 10°C as described for panel B, lane 3. Samples of the crosslinking reactions (2 µg) were mixed with 5 × SDS sample-loading buffer devoid of DTT, and separated on SDS-PAGE. <b>D,</b> The effect of K357A or R260A mutation on disulfide formation between I34C and V240C. The crosslinking was performed as described in panel B for 5 min at 10°C. Equal loading of lanes was confirmed by densitometry of the same samples on Coomassie-stained SDS-PAGE (not shown).