LOCALIZATION OF DEUTERIUM LABELS IN PURPLE MEMBRANES BY NEUTRON DIFFRACTION: POWER AND LIMITATIONS OF THE METHOD

Martin Weik^* and Giuseppe Zaccai

Institut de Biologie Structurale, 41, Avenue des Martyrs, 38027 Grenoble Cedex 1, France

* present address: Bijvoet Center for Biomolecular Research, Padualaan 8, 3584 CH Utrecht, The Netherlands

The purple membrane (PM) forms crystalline patches in the cytoplasmic membrane of the archaeon Halobacterium salinarum. It is made up of bacteriorhodopsin (BR), a light-activated proton pump, and various lipid species, i.e. phospho- and glycolipids. Because of its remarkable crystallinity, the PM is, so far, the only natural membrane whose structure can be examined directly by diffraction methods at sub-molecular resolution.

Due to the pronounced difference in coherent neutron scattering lengths of hydrogen and deuterium atoms, neutron diffraction is a unique method to localize specifically 2H-labeled components and exchangeable water molecules in a crystalline sample. Applying the difference Fourier method allows not only to localize labels but also to estimate their strength. In that context it is important to note that isotopic H/²H exchange does not alter the structure under study.

The power and limitations of the method are discussed with respect to two structural studies of PM that contributed to the understanding of its function; the structure and hydration of an intermediate state of the BR reaction cycle [1] and the localization of the PM glycolipids and their interaction with BR [2]. The first study is an example where the relative strengths of difference Fourier peaks had to be determined accurately, whereas in the second study only the distribution of low resolution difference density was interpreted.

[1] Weik M., Zaccai G., Dencher N., Oesterhelt D. and Hauss T. (1998). Structure and Hydration of the M-state of the Bacteriorhodopsin Mutant D96N Studied by Neutron Diffraction. J. Mol. Biol. 275, 625-634.

[2] Weik M., Patzelt H., Zaccai G. and Oesterhelt D. (1998). Localization of Glycolipids in Membranes by In Vivo Labeling and Neutron Diffraction. Mol. Cell. 1, 411-419.