SITE-DIRECTED SPIN-LABELING AND CHEMICAL CROSS-LINKING DEMONSTRATE THAT HELIX-V IS CLOSE TO HELIX-VII AND HELIX-VIII IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

Site-directed chemical cleavage of lactose permease indicates that hel ix V is in close proximity to helices VII and VIII. To test this concl usion further, permease containing a biotin-acceptor domain and paired Cys residues at positions 148 (helix V) and 228 (helix VII), 148 and 226 (helix VII), or 148 and 275 (helix VIII) was affinity purified and labeled with a sulfhydryl-specific nitroxide spin label. Spin-spin in teractions are observed with the 148/228 and 148/275 pairs, indicating close proximity between appropriate faces of helix V and helices VII and VIII. Little or no interaction is evident with the 148/226 pair, i n all likelihood because position 226 is on the opposite face of helix VII from position 228. Broadening of the electron paramagnetic resona nce spectra in the frozen state was used to estimate distance between the 148/228 and the 148/275 pairs. The nitroxides at positions 148 and 228 or 148 and 275 are within approximate to 13-15 Angstrom. Finally, Cys residues at positions 148 and 228 are crosslinked by dibromobiman e, a bifunctional crosslinker that is approximate to 5 Angstrom long, while no crosslinking is detected between Cys residues at positions 14 8 and 275 or 148 and 226, The results provide strong support for a str ucture in which helix V is in close proximity to both helices VII and VIII and is oriented in such a fashion that Cys-148 is closer to helix VII.