¨Hydrophobic Photolabelling Analysis of Lipid-Protein Stoichiometry.

VILLAMIL GIRALDO, A. M.; CASTELLO, P. R.; GONZÁLEZ FLECHA, F.L.; DELFINO, J.M.; ROSSI, J.P.F.C.

Montpellier, Francia

Congreso; 15th IUPAB & 5th EBSA International Biophysics Congress; 2005

IUPAB

Introduction   Plasma membrane calcium pump is an integral membrane protein that actively transports Ca2+ across the cellular membrane. The enzyme is comprised of a single chain of 134 kDa in which ten membrane regions are predicted by hydropathy plots (MPEX White). As many other membrane proteins it is particularly sensitive to its phospholipid environment (CITAS). Both the enzyme activity and its response to different modulators are highly dependent on its interaction with membrane phospholipids. (Niggli et al., 1981) Regarding the enzymatic activity of PMCA, neutral phospholipids play a general role i.e., they do not fully activate the enzyme but are required to maintain the protein in such a conformation that it can be further activated (Gazzotti et al., 1994). Hydrophobic photolabelling techniques have proved to be useful for identifying protein domains which are in direct contact with membrane phospholipids. Using these techniques three hydrophobic domains have been identified in PMCA (Castello et al, 1994). In this work we have employed a phosphatidylcholine analogue [125I] TID-PC/16 that has been previously used to analyse lipid protein interfaces ( Córsico et al 2000, Blanton & Mc. Cardy 2000). Our aim was to obtain further structural information about membrane regions of PMCA and their interaction with surrounding lipids. To this end we have assessed the number of phospholipid molecules directly lining the transmembrane surface and evaluated the distribution of these phospholipid molecules around the intramembraneous region.