Alteration of Substrate-Induced Conformational Changes ofEscherichia coliMelibiose Permease by Mutating Arg149

Abstract

AbstractFourier transform infrared difference spectroscopy and fluorescence spectroscopic techniques have been used to obtain information about substrate-induced structural changes of the melibiose permease mutant R149C, compared with the Cys-less, which were reconstituted into liposomes. ATR-FTIR evidences show that Na+-induced difference spectra of R149C and Cys-less are similar. However, Na+induces some new peaks for R149C mutant permease. This means that replacement of Arg-149 by Cys may affect the structure of MelB, and then affect the binding of Na+. Melibiose-induced difference spectra of R149C in the presence of Na+show some peaks in the amide I region not seen in Cys-less, corresponding to turns, β-sheets, α-helix changes. This suggests that R149C mutant permease undergo some different secondary structure changes compared to Cys-less mutant permease, when binding melibiose. Comparison of the permease intrinsic fluorescence variations of R149C and Cys-less indicate that there are similar substrate binding properties between R149C and Cys-less. When analyzing the effects of different sugars it appears that the R149C mutant is more sensitive to the sugar. All these data indicate that replacement of Arg-149 by Cys will affect Na+and sugar binding, and enhance the selectivity and sensitivity to sugars.