MID-MEMBRANE PHOTOLABELING OF THE TRANSMEMBRANE DOMAIN OF GLYCOPHORIN A IN PHOSPHOLIPID VESICLES
Laboratoire de Chimie Organique des Substances Naturelles, associé au CNRS, Université Louis Pasteur, 5 rue Blaise Pascal, F-67084 Strasbourg
It is very important to obtain by novel methods direct evidence on the topography of proteins in membranes. Khorana and later Breslow and Schreiber have tried to use, for this goal, amphiphilic photosensitive probes, in the hope that irradiation of reconstituted membranes containing them and a protein would lead to labeling at a definite level inside the bilayer. However, their results have been uniformly disappointing, as the labeling is not at all specific, due certainly to the extensive dynamics of the phospholipidic matrix and of the probe itself.
We have previously developed a novel type of a bola-amphiphilic probe, which has been shown to be remarkably selective for attack of lipid membrane components near the middle of the bilayer, due to its transmembrane construction and to the concomitant use of cholesterol to improve the order of the lipidic chains of the membrane.
In this communication we show the use of this method to the study of the topography of the membrane domain of glycophorin A, the amino acid sequence of which was the first primary structure of a membrane protein ever elucidated. For the first time, to our knowledge, the center of the transmembrane domain of a protein has been selectively functionalized in membranes.
Key References :
- Nakatani, Y.; Yamamoto, M.; Diyizou, Y.; Warnock, W.; Dollé, V.; Hahn, W.; Milon, A.; Ourisson, G. : $B!H(BStudies on the topography of biomembranes : regioselective photolabelling in vesicles with the tandem use of cholesterol and a photoactivatable transmembrane probe$B!I(B, Chem. Eur. J. 1996, 2, 129-138.
- Deseke, E., Nakatani, Y., Ourisson, G. : "Intrinsic reactivities of amino acids towards photoalkylation with benzophenone- a study preliminary to photolabelling of the transmembrane protein glycophorin A", Eur. J. Org. Chem. 1998, 243-251.
- Ogawa, Y., Hahn, W., Garnier, P., Higashi, N., Massotte, D., Metz-Boutigue, M. H., Rousseau, B., Sunamoto, J., Ourisson, G., Nakatani, Y. : "Mid-membrane photolabeling of the transmembrane domain of glycophorin A in phospholipid vesicles", Angew. Chem. Int. Ed. Engl., 2001, 40, 944-946.