FTIR Spectroscopy of Secondary-Structure Reorientation of Melibiose Permease Modulated by Substrate Binding

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Analysis of infrared polarized absorbance spectra and linear dichroism spectra of reconstituted melibiose permease from Escherichia coli shows that the oriented structures correspond mainly to tilted transmembrane α-helices, forming an average angle of ∼26° with the membrane normal in substrate-free medium. Examination of the deconvoluted linear dichroism spectra in H 2O and D 2O makes apparent two populations of α-helices differing by their tilt angle (helix types I and II). Moreover, the average helical tilt angle significantly varies upon substrate binding: it is increased upon Na + binding, whereas it decreases upon subsequent melibiose binding in the presence of Na +. In contrast, melibiose binding in the presence of H + causes virtually no change in the average tilt angle. The data also suggest that the two helix populations change their tilting and H/D exchange level in different ways depending on the bound substrate(s). Notably, cation binding essentially influences type I helices, whereas melibiose binding modifies the tilting of both helix populations.