HELIX PACKING OF LACTOSE PERMEASE IN ESCHERICHIA-COLI STUDIED BY SITE-DIRECTED CHEMICAL CLEAVAGE

Biotinylated lactose permease from Escherichia coli containing a singl e-cysteine residue at position 330 (helix X) or at position 147, 148, or 149 (helix V) was purified by avidin-affinity chromatography and de rivatized with -(alpha-bromoacetamido)-1,10-phenanthroline-copper [OP( Cu)], Studies with purified, OP(Cu)-labeled Leu-330 --> Cys permease i n dodecyl-beta-D-maltopyranoside demonstrate that after incubation in the presence of ascorbate, cleavage products of approximate to 19 and 6-8 kDa are observed on immunoblots with anti-C-terminal antibody, Rem arkably, the same cleavage products are observed with permease embedde d in the native membrane, Comparison with the C-terminal half of the p ermease expressed independently as a standard indicates that the 19-kD a product results from cleavage near the cytoplasmic end of helix VII, whereas the 6- to 8-kDa fragment probably results from fragmentation near the cytoplasmic end of helix XI, Results are entirely consistent with a tertiary-structure model of the C-terminal half of the permease derived from earlier site-directed fluorescence and site-directed mut agenesis studies, Similar studies with OP(Cu)-labeled Cys-148 permease exhibit cleavage products at approximate to 19 kDa and at 15-16 kDa, The larger fragment probably reflects cleavage at a site near the cyto plasmic end of helix VII, whereas the 15- to 16-kDa fragment is consis tent with cleavage near the cytoplasmic end of helix VIII, When OP(Cu) is moved 100 degrees to position 149 (Val-149 --> Cys permease), a si ngle product is observed at 19 kDa, suggesting fragmentation al the cy toplasmic end of helix VII, However, when the reagent is moved 100 deg rees in the other direction to position 147 (Gly-147 --> Cys permease) , cleavage is not observed, The results suggest that helix V is in clo se proximity to helices VII and VIII with position 148 in the interfac e between the helices, position 149 facing helix VII, and position 147 facing the lipid bilayer.