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

Natàlia Dave ^*, Víctor A. Lórenz-Fonfría ^*, Gérard Leblanc  and Esteve Padrós ^*

^* Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, and Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, Barcelona, Spain; and Institut de Biologie et Technologies-Saclay, Service de Bioenergétique, Biologie Structurale et Mécanismes, Gif sur Yvette, France

Correspondence: Address reprint requests to Natàlia Dave, Unitat de Biologia Cellular i Molecular, Institut Municipal d'Investigació Mèdica, Universitat Pompeu Fabra, Barcelona, Spain. E-mail: ndave{at}imim.es.

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 -helices, forming an average angle of 26° with the membrane normal in substrate-free medium. Examination of the deconvoluted linear dichroism spectra in H₂O and D₂O makes apparent two populations of -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.