Estudio de las interacciones Lípido-Proteína utilizando un reactivo fotoactivable

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

Mar Del Plata

Congreso; I Congreso Conjunto de Sociedades Biomédicas; 2004

Sociedades Biomédicas

INTERACTIONS BETWEEN PLASMA MEMBRANE CALCIUM PUMP AND PHOSPHOLIPIDS STUDIED WITH A PHOTOACTIVATABLE PROBE   Villamil Giraldo, A.M., Castello, P.R., Delfino, J.M, González Flecha, F.L. and Rossi, J.P.F.C. IQUIFIB. and F. F. y Bioquímica, U.B.A. Junín 956. Buenos Aires, Argentina. anamaria@qb.ffyb.uba.ar   The function of membrane proteins are highly dependent on their phospholipid environment. In order to study the interaction between Plasma Membrane Calcium Pump (PMCA) and phospholipids we have used the phosphatidylcholine photoactivatable analogue ([125I] TID-PC/16) and a purified solubilized preparation of PMCA. With this approach, we analysed the lipid annulus around the transmembrane domain determining the number of lipid molecules that are in close contact with the transmembrane surface and the distribution of these lipid molecules within the transmembrane domains of the protein. PMCA was labeled in the presence of different amounts of phosphatidylcholine (PC). Lipid-protein stoichiometry was determined by evaluating the extent of the labeling reaction. The number of PC molecules in direct contact with the protein follows a hyperbolic function of the concentration of PC . The maximum number of PC molecules that are in direct contact with the transmembrane surface is 17 ± 1 PC/protein. With the aim of evaluating its general applicability we assayed this analytical method on Sarcoplasmic Reticulum Calcium Pump. Lipid stoichiometry was previously measured for this protein using spin-label electron paramagnetic resonance (EPR) techniques and the values obtained were between 22±2 and 24±5 phospholipids/monomer (Silvius et al. Biochemistry. 1984, 23:538-47; Thomas et al. Biophys J. 1982 37:217-25. ) According to our results 19 ± 3 molecules of PC would be surrounding the transmembrane surface of this protein. Our experimental approach could serve as a good alternative when as in the case of PMCA, protein concentration is too low to apply EPR techniques or, when more than one membrane protein is simultaneously present. A qualitative analysis of the interaction of PMCA with its phospholipid environment was performed through limited proteolysis of labeled PMCA and further analysis of the specific incorporation of the probe to each fragment. According to our results, N-terminal and central regions, both containing two transmembrane segments, would differentially interact with membrane phospholipids. In addition, only four of the six transmembrane segments present in the C-terminal region of the protein would be exposed to membrane lipids. This relative distribution of phospholipids around the transmembrane surface of the protein does not depend on the concentration of PC.