FTIR spectroscopy of secondary structure re-orientation of melibiose permease modulated by substrate binding

¹ Unitat de Biofisica. Departament de Bioquimica i Biologia Molecular. Universitat Autonoma Barcelona
² Unitat de Biofisica. Departament de Bioquimica i Biologia Molecular. Universitat Autonma Barcelona
³ Institute de Biologie et Technologies-Saclay,Service de Bioenergetique.

^* To whom correspondence should be addressed. E-mail: ndave{at}imim.es.

Submitted on June 20, 2007
Revised on July 16, 2007
Accepted on 24 October 2007

	Abstract

Analysis of infrared polarized absorbance spectra and linear dichroism spectra of reconstituted melibiose permease from E. coli shows that the oriented structures correspond mainly to tilted transmembrane helices, forming an average angle of about 26° with the membrane normal in substrate-free medium. Examination of the deconvoluted linear dichroism spectra in H₂O and in D₂O makes apparent two populations of helices differing by their tilt angle (helices types I and II). Moreover, the average helical tilt angle significantly varies upon substrate binding: it is increased upon Na⁺ binding, while 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.

Key Words: ATR-FTIR, MelB, alfa-helices, membrane transporter, polarization, secondary structure orientation