THE LACTOSE PERMEASE MEETS FRANKENSTEIN

The lactose permease (lac) of Escherichia coli is a paradigm for membr ane transport proteins. Encoded by the lacY gene, the permease has bee n solubilized, purified to homogeneity, reconstituted into phospholipi d vesicles and shown to catalyse the coupled translocation of beta-gra lactosides and HC with a stoichiometry of unity. Circular dichroism an d other spectroscopic approaches demonstrate that the purified permeas e is about 80 % helical. Based on hydropathy analysis of the primary a mino-acid sequence, a secondary structure has been proposed in which t he protein has 12 hydrophobic domains in alpha-helical conformation th at traverse the membrane in zigzag fashion connected by hydrophilic lo ops. A variety of other approaches are consistent with the model and d emonstrate that both the N and C termini are on the inner surface of t he membrane, and studies on an extensive series of lac permease/alkali ne phosphatase fusion proteins provide exclusive support for the topol ogical predictions of the 12-helix motif. This presentation concentrat es on the use of site-directed fluorescence spectroscopy to study stru cture-function relationships in the permease.