![Sample our Bioscience journals, sign in here to start your access, Latest two full volumes FREE to you for 14 days]({"type":"image" , "src" : "/sda/5925/TOC-Subject-Sample-2021-Bioscience.jpg"})
Abstract
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 coli. By taking advantage of the finding that labelling of single-Cys148 permease with the thiol-specific fluorophore 2-(4-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) is blocked specifically by substrates of the permease, it is demonstrated that the high-affinity ligand β, D-galactopyrano-syl 1-thio-β, D-galactopyranoside (TDG) stabilizes solu-biked, puirified permease against heat denaturation. Furthermore, TDG protection against MIANS labelling of single-Cys148 permease is abolished by guanidinium hydrochloride. After dialysis of the denaturant, TDG protection against MIANS labelling is recovered, indicating that the permease has been refolded. The conclusion is confirmed and extended by studying site-directed fluorescence of purified single-Cys331 permease, where the emission spectrum of the MIANS-labelled protein is differentially altered by low or high concentrations of TDG. The results demonstrate that both low -and high-affinity binding, as well as ligand-induced conformational changes in the permease, can be denatured reversibly in vitro.