In-plane and out-of-plane infrared difference spectroscopy unravels tilting of helices and structural changes in a membrane protein upon substrate binding

(Chemical Equation Presented) Attenuated total reflection infrared (ATR-IR) difference spectroscopy stands out because of its ability to provide information on the interaction of substrates with membrane proteins in their native lipid bilayer environment. We show how the study and interpretation of the structural changes in membrane proteins upon substrate binding is simplified by obtaining ATR-IR difference spectra with polarized light and then computing the difference spectra in the z and x,y directions, where structural and orientation changes give specific difference absorbance patterns. In combination with a maximum-entropy band-narrowing method and some simple spectroscopic rules, the present approach allows us to unambiguously identify changes in the tilt of some helices in the secondary transporter melibiose permease following melibiose binding in the presence of sodium, suggesting the formation of an occluded state during the transport mechanism of the substrates. © 2009 American Chemical Society.