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Evaluation of the Ordering of Membranes in Multilayer Stacks Built on an ATR-FTIR Germanium Crystal with Atomic Force Microscopy: The Case of the H⁺,K⁺-ATPase-containing Gastric Tubulovesicle Membranes

Dimitri Ivanov ^*, Nicolas Dubreuil ^*, Vincent Raussens , Jean-Marie Ruysschaert  and Erik Goormaghtigh 

^* Laboratory for Polymer Physics, Laboratory for the Structure and Function of Biological Membranes, Structural Biology and Bioinformatics Center, Free University of Brussels, Brussels, Belgium

Correspondence: Address reprint requests to Dr. E. Goormaghtigh, Laboratory of Structure and Function of Biological Membranes, Université Libre de Bruxelles, CP 206/2, Boulevard du Triomphe, B-1050 Brussels, Belgium. Tel.: 32-2-650-5386; Fax: 32-2-650-5382; E-mail: egoor{at}ulb.ac.be.

Polarized attenuated total reflection Fourier transform infrared spectra were recorded on multilayer stacks of native gastric tubulovesicle membranes. The spectral intensity and linear dichroism were measured for average thicknesses ranging between 0 and 100 bilayers. Atomic force microscopy was used to investigate the orientation of the membranes at the top of the stack. Height profiles were obtained along randomly drawn lines and slopes were computed over various distances. Orientation distribution functions were obtained from the slopes and decomposed into Legendre polynomials. It was found that the second Legendre polynomials coefficient characterizing the membrane orientation was always larger than 0.9. It could therefore be concluded that the membrane tilt does not significantly contribute to the infrared dichroism, even for the largest thicknesses tested.

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