SITE-DIRECTED SPIN-LABELING DEMONSTRATES THAT TRANSMEMBRANE DOMAIN-XII IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI IS AN ALPHA-HELIX

Functional lactose permease mutants containing single-Cys residues at positions 387-402 [He, M. M., Sun, J., & Kaback, H. R. (1996) Biochemi stry 35, 12909-12914] and a biotin acceptor domain in the middle cytop lasmic loop were solubilized in n-dodecyl-beta-D-maltopyranoside and p urified by avidin affinity chromatography. Each mutant protein was der ivatized with a thiol-selective nitroxide reagent and examined by conv entional and power saturation electron paramagnetic resonance spectros copy. Analysis of the electron paramagnetic resonance spectral line sh apes and the influence of O-2 On the saturation behavior of the spin-l abeled proteins were measured in order to obtain information on the mo bility of the spin-labeled side chains and their accessibility to O-2, respectively. The data show a periodic dependence of both mobility an d accessibility on sequence position consistent with an alpha-helical structure. These results provide direct support for the contention tha t transmembrane domain XII is in an alpha-helical conformation and on the periphery of the 12-helix bundle that comprises the lactose permea se molecule.