HELIX PROXIMITY AND LIGAND-INDUCED CONFORMATIONAL-CHANGES IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI DETERMINED BY SITE-DIRECTED CHEMICAL CROSS-LINKING

N and C-terminal halves of lactose permease, each with a single-Cys re sidue, were co-expressed, and crosslinking was studied. Iodine or N,N' -o-phenylenedimaleimide (o-PDM; rigid 6 Angstrom), crosslinks Asn245 - -> Cys (helix VII) and Il52 --> Cys or Ser53 --> Cys (helix II). N,N'- p-phenylenedimaleimide (p-PDM; rigid 10 Angstrom) crosslinks the 245/5 3. Cys pair weakly, but does not crosslink 245/52, and 1,6-bis-maleimi dohexane (BMH; flexible 16 Angstrom) crosslinks both pairs less effect ively than o-PDM. Thus, 245 is almost equidistant from 52 and 53 by up to about 6 Angstrom. BMH or p-PDM crosslinks Gln242 --> Cys and Ser53 --> Cys, but o-PDM is ineffective, indicating that distance varies by up to 10 A. Ligand binding increases crosslinking of 245/53 with p-PD M or BMH, has little effect with o-PDM and decreases iodine crosslinki ng. Similar effects are observed with 245/52. Ligand increases 242/53 crosslinking with p-PDM or BMH, but no crosslinking is observed with o -PDM. Therefore, ligand induces a translational or scissors-like displ acement of the helices by 3-4 Angstrom. Crosslinking 245/53 inhibits t ransport indicating that conformational flexibility is important for f unction. (C) 1997 Academic Press Limited.