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

Plasmids encoding "split" lactose permease constructs with discontinuities in either the periplasmic loop between helices V and VI (N-5/C-7) or between helices VI and VII (N-6/C-6) were used to localize helix VI within the tertiary 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 cross-linking is observed between the periplasmic ends of helices V (position 158 or 161) and VI (position 170) with rigid 6 or 10 Angstrom reagents. Furthermore, the Cys residue at position 170 (helix VI) also cross-links to a Cys residue at either position 264 or 265 (helix VIII) with a 21 Angstrom cross-linking agent. The data indicate that helices V, VI and VIII are in close proximity at the periplasmic face of the membrane, with helix VI significantly closer to helix V. In addition, beta ,D-galactopyranosyl 1-thio-beta ,D-galactopyranoside induces a significant increase in cross-linking efficiency between helices VI and VIII and between helices V and VIII, with no significant change in cross-linking between helices V and VI. (C) 2001 Academic Press.