Helix packing in the lactose permease of Escherichia coli: Localization ofhelix VI

Plasmids encoding "split" lactose permease constructs with discontinuities in either the periplasmic loop between helices V and VI (N-5/C-7) or betwee n helices VI and VII (N-6/C-6) were used to localize helix VI within the te rtiary structure by site-directed thiol cross-linking. A total of 57 double -Cys pairs, with one Cys residue in helix VI and another in helix V or VIII , were studied with homobifunctional cross-linking agents. Significant cros s-linking is observed between the periplasmic ends of helices V (position 1 58 or 161) and VI (position 170) with rigid 6 or 10 Angstrom reagents. Furt hermore, the Cys residue at position 170 (helix VI) also cross-links to a C ys residue at either position 264 or 265 (helix VIII) with a 21 Angstrom cr oss-linking agent. The data indicate that helices V, VI and VIII are in clo se proximity at the periplasmic face of the membrane, with helix VI signifi cantly closer to helix V. In addition, beta ,D-galactopyranosyl 1-thio-beta ,D-galactopyranoside induces a significant increase in cross-linking effic iency between helices VI and VIII and between helices V and VIII, with no s ignificant change in cross-linking between helices V and VI. (C) 2001 Acade mic Press.