FIG 2.

Membrane topology of ArgO. (A) Two-dimensional topological model of ArgO. The lengths of the TM segments were set to those obtained for the in silico model of ArgO described in Fig. 9. The open and closed circles represent, respectively, the inferred positions of the Cys substitutions engineered into Cys-less ArgO and two of the three naturally occurring Cys residues, Cys43 and Cys52, in ArgO. The positions of PhoA fusions to ArgO are marked with triangles, and the HA tag is shown. (B) Cys accessibility depicting immunodetection of the ArgO–MAL-PEG adducts. Cultures of GJ9099.1 containing plasmids encoding the single-Cys-substituted ArgO proteins indicated were processed, and the ArgO–MAL-PEG (Ia-VIIa) and H-NS–MAL-PEG (Ib-VIIb) adducts were detected by Western blotting with anti-HA and anti-FLAG antibodies, respectively. U, T, and S stand for samples that were not treated with MAL-PEG, treated with MAL-PEG, and treated with MAL-PEG following sonication, respectively. The ArgO–MAL-PEG and H-NS–MAL-PEG adducts and free ArgO and H-NS species are marked with filled and open triangles, respectively.