IN-VITRO FOLDING OF A MEMBRANE-PROTEIN - EFFECT OF DENATURATION AND RENATURATION ON SUBSTRATE-BINDING BY THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

Site-directed mutagenesis and site-directed fluorescence spectroscopy demonstrate that Cys148 interacts hydrophobically with the galactosyl moiety of substrates of the lactose permease of Escherichia coil. By t aking advantage of the finding that labelling of single-Cys148 permeas e with the thiol-specific fluorophore 2-(4'-maleimidylanilino)naphthal ene-6-sulfonic acid (MIANS) is blocked specifically by substrates of t he permease, it is demonstrated that the high-affinity ligand beta,D-g alactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) stabilizes solu- bilized, purified permease against heat denaturation. Furthermore, TD G protection against MIANS labelling of single-Cys148 permease is abol ished by guanidinium hydrochloride. After dialysis of the denaturant, TDG protection against MIANS labelling is recovered, indicating that t he permease has been refolded, The conclusion is confirmed and extende d by studying site-directed fluorescence of purified single-Cys331 per mease, where the emission spectrum of the MIANS-labelled protein is di fferentially altered by low or high concentrations of TDG. The results demonstrate that both low-and high-affinity binding, as well as ligan d-induced conformational changes in the permease, can be denatured rev ersibly in vitro.