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Lipid-protein interactions play an essential role in the regulation of biological function of integral membrane proteins; however, the underlying molecular mechanisms are not fully understood. Here we explore the modulation by phospholipids of the enzymatic activity of the plasma membrane calcium pump reconstituted in detergent-phospholipid mixed micelles of variable composition. The presence of increasing quantities of phospholipids in the micelles produced a cooperative increase in the ATPase activity of the enzyme. This activation effect was reversible and depended on the phospholipid/detergent ratio and not on the total lipid concentration. Enzyme activation was accompanied by a small structural change at the transmembrane domain reported by 1-aniline-8-naphtalenesulfonate fluorescence. In addition, the composition of the amphipilic environment sensed by the protein was evaluated by measuring the relative affinity of the assayed phospholipid for the transmembrane surface of the protein. The obtained results allow us to postulate a two-stage mechanistic model explaining the modulation of protein activity based on the exchange among non-structural amphiphiles at the hydrophobic transmembrane surface, and a lipid-induced conformational change. The model allowed to obtain a cooperativity coefficient reporting on the efficiency of the transduction step between lipid adsorption and catalytic site activation. This model can be easily applied to other phospholipid/detergent mixtures as well to other membrane proteins. The systematic quantitative evaluation of these systems could contribute to gain insight into the structure-activity relationships between proteins and lipids in biological membranes. © 2012 Dodes Traian et al.


Documento: Artículo
Título:A two-stage model for lipid modulation of the activity of integral membrane proteins
Autor:Dodes Traian, M.M.; Cattoni, D.I.; Levi, V.; González Flecha, F.L.
Filiación:Laboratorio de Biofísica Molecular, Instituto de Química y Fisicoquímica Biológicas, Universidad de Buenos Aires - CONICET, Buenos Aires, Argentina
Laboratorio de Dinámica Intracelular- Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
Centre de Biochimie Structurale, INSERM U554, CNRS UMR 5048, Université de Montpellier 1 and 2, Montpellier, France
Palabras clave:8 anilino 1 naphthalenesulfonic acid; adenosine triphosphatase (calcium); amphophile; membrane protein; phospholipid; adsorption kinetics; article; binding affinity; conformational transition; controlled study; enzyme activation; enzyme active site; enzyme activity; enzyme structure; fluorescence spectroscopy; human; human cell; hydrophobicity; micelle; molecular mechanics; molecular model; protein domain; protein lipid interaction; signal transduction; Algorithms; Enzyme Activation; Humans; Membrane Proteins; Micelles; Models, Molecular; Phospholipids; Plasma Membrane Calcium-Transporting ATPases
Título revista:PLoS ONE
Título revista abreviado:PLoS ONE
CAS:8 anilino 1 naphthalenesulfonic acid, 82-76-8; Membrane Proteins; Micelles; Phospholipids; Plasma Membrane Calcium-Transporting ATPases,


  • Singer, S.J., Nicolson, G.L., The fluid mosaic model of the structure of cell membranes (1972) Science, 175, pp. 720-731
  • Phillips, R., Ursell, T., Wiggins, P., Sens, P., Emerging roles for lipids in shaping membrane-protein function (2009) Nature, 459, pp. 379-385
  • Lee, A.G., Biological membranes: the importance of molecular detail (2011) Trends in Biochemical Sciences, 36, pp. 493-500
  • Adamian, L., Naveed, H., Liang, J., Lipid-binding surfaces of membrane proteins: Evidence from evolutionary and structural analysis (2011) Biochim Biophys Acta, 1808, pp. 1092-1102
  • Hunte, C., Richers, S., Lipids and membrane protein structures (2008) Curr Op Struct Biol, 18, pp. 406-411
  • Jost, P.C., Griffith, O.H., Capaldi, R.A., Vanderkooi, G., Evidence for boundary lipid in membranes (1973) Proc Nat Acad Sci USA, 70, pp. 480-484
  • Marsh, D., Electron spin resonance in membrane research: Protein-lipid interactions (2008) Methods, 46, pp. 83-96
  • Jost, P.C., Griffith, O.H., The lipid-protein interface in biological membranes (1980) Ann N Y Acad Sci, 348, pp. 391-407
  • Lee, A.G., How lipids affect the activities of integral membrane proteins (2004) Biochim Biophys Acta, 1666, pp. 62-87
  • Strehler, E.E., James, P., Fischer, R., Heim, R., Vorherr, T., Peptide sequence analysis and molecular cloning reveal two calcium pump isoforms in the human erythrocyte membrane (1990) J Biol Chem, 265, pp. 2835-2842
  • Di Leva, F., Domi, T., Fedrizzi, L., Lim, D., Carafoli, E., The plasma membrane Ca2++ ATPase of animal cells: structure, function and regulation (2008) Arch Biochem Biophys, 476, pp. 65-74
  • Castello, P.R., Caride, A.J., González Flecha, F.L., Fernández, H.N., Rossi, J.P., Identification of transmembrane domains of the red cell calcium pump with a new photoactivatable phospholipidic probe (1994) Biochem Biophys Res Commun, 201, pp. 194-200
  • Castello, P.R., González Flecha, F.L., Caride, A.J., Fernández, H.N., Delfino, J.M., The membrane topology of the amino-terminal domain of the red cell calcium pump (1997) Protein Sci, 6, pp. 1708-1717
  • Kosk-Kosicka, D., Comparison of the red blood cell Ca2++-ATPase in ghost membranes and after purification (1990) Mol Cell Biochem, 99, pp. 75-81
  • Carafoli, E., Biogenesis: plasma membrane calcium ATPase: 15 years of work on the purified enzyme (1994) FASEB J, 8, pp. 993-1002
  • Levi, V., Rossi, J.P., Echarte, M.M., Castello, P.R., González Flecha, F.L., Thermal stability of the plasma membrane calcium pump. Quantitative analysis of its dependence on lipid-protein interactions (2000) J Membr Biol, 173, pp. 215-225
  • Tang, D., Dean, W.L., Borchman, D., Paterson, C.A., The influence of membrane lipid structure on plasma membrane Ca2++ -ATPase activity (2006) Cell Calcium, 39, pp. 209-216
  • Ronner, P., Gazzotti, P., Carafoli, E., A lipid requirement for the (Ca2++Mg2+)-activated ATPase of erythrocyte membranes (1977) Arch Biochem Biophys, 179, pp. 578-583
  • Niggli, V., Adunyah, E.S., Carafoli, E., Acidic phospholipids, unsaturated fatty acids, and limited proteolysis mimic the effect of calmodulin on the purified erythrocyte Ca2++ - ATPase (1981) J Biol Chem, 256, pp. 8588-8592
  • Filomatori, C.V., Rega, A.F., On the mechanism of activation of the plasma membrane Ca2++-ATPase by ATP and acidic phospholipids (2003) J Biol Chem, 278, pp. 22265-22271
  • Garber Cohen, I.P., Castello, P.R., González Flecha, F.L., Ice-induced partial unfolding and aggregation of an integral membrane protein (2010) Biochim Biophys Acta, 1798, pp. 2040-2047
  • Levi, V., Rossi, J.P., Castello, P.R., González Flecha, F.L., Structural significance of the plasma membrane calcium pump oligomerization (2002) Biophys J, 82, pp. 437-446
  • Tanford, C., (1980) The Hydrophobic Effect, p. 234. , John Wiley & Sons
  • Ladokhin, A.S., Fernández-Vidal, M., White, S.H., CD spectroscopy of peptides and proteins bound to large unilamellar vesicles (2010) J Membr Biol, 236, pp. 247-253
  • Cattoni, D.I., González Flecha, F.L., Argüello, J.M., Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus (2008) Arch Biochem Biophys, 471, pp. 198-206
  • Roman, E.A., Santos, J., González Flecha, F.L., The use of circular dichroism methods to monitor unfolding transitions in peptides, globular and membrane proteins (2012), pp. 217-254. , In: Rodgers DS, editors, editor; Yau, W.M., Wimley, W.C., Gawrisch, K., White, S.H., The preference of tryptophan for membrane interfaces (1998) Biochemistry, 37, pp. 14713-14718
  • Levi, V., Rossi, J.P.F.C., Castello, P.R., González Flecha, F.L., Oligomerization of the plasma membrane calcium pump involves two regions with different thermal stability (2000) FEBS Letters, 483, pp. 99-103
  • Kozachkov, L., Padan, E., Site-directed tryptophan fluorescence reveals two essential conformational changes in the Na+/H+ antiporter NhaA (2011) Proc Nat Acad Sci USA, 108, pp. 15769-15774
  • Cattoni, D.I., Kaufman, S.B., González Flecha, F.L., Kinetics and thermodynamics of the interaction of 1-anilino-naphthalene-8-sulfonate with proteins (2009) Biochim Biophys Acta, 1794, pp. 1700-1708
  • Daniel, E., Weber, G., Cooperative effects in binding by bovine serum albumin. I. The binding of 1-anilino-8-naphthalenesulfonate. Fluorimetric titrations (1966) Biochemistry, 5, pp. 1893-1900
  • Roman, E.A., Argüello, J.M., González Flecha, F.L., Reversible unfolding of a thermophilic membrane protein in phospholipid/detergent mixed micelles (2010) J Mol Biol, 397, pp. 550-559
  • Marsh, D., Protein modulation of lipids, and vice-versa, in membranes (2008) Biochim Biophys Acta, 1778, pp. 1545-1575
  • Atkins, P., de Paula, J., (2006) Physical Chemistry, , New York: WH Freeman and Company
  • Levi, V., Rossi, J.P., Castello, P.R., González Flecha, F.L., Quantitative analysis of membrane protein-amphiphile interactions using resonance energy transfer (2003) Anal Biochem, 317, pp. 171-179
  • Loura, L.M., Prieto, M., Fernandes, F., Quantification of protein-lipid selectivity using FRET (2010) Eur Biophys J, 39, pp. 565-578
  • Reed, L.J., Berkson, J., The application of the logistic function to experimental data (1928) J Phys Chem, 33, pp. 760-779
  • O'Keeffe, A.H., East, J.M., Lee, A.G., Selectivity in lipid binding to the bacterial outer membrane protein OmpF (2000) Biophys J, 79, pp. 2066-2074
  • Sanders, C.R., Mittendorf, K.F., Tolerance to changes in membrane lipid composition as a selected trait of membrane proteins (2011) Biochemistry, 50, pp. 7858-7867
  • Walsh, J.P., Bell, R.M., sn-1,2-Diacylglycerol kinase of Escherichia coli. Structural and kinetic analysis of the lipid cofactor dependence (1986) J Biol Chem, 261, pp. 15062-15069
  • González Flecha, F.L., Castello, P.R., Gagliardino, J.J., Rossi, J.P., Molecular characterization of the glycated plasma membrane calcium pump (1999) J Membr Biol, 171, pp. 25-34
  • Levi, V., Villamil Giraldo, A.M., Castello, P.R., Rossi, J.P., González Flecha, F.L., Effects of phosphatidylethanolamine glycation on lipid-protein interactions and membrane protein thermal stability (2008) Biochem J, 416, pp. 145-152
  • Schägger, H., Tricine-SDS-PAGE (2006) Nat Protoc, 1, pp. 16-22
  • Strop, P., Brunger, A.T., Refractive index-based determination of detergent concentration and its application to the study of membrane proteins (2005) Protein Sci, 14, pp. 2207-2211
  • Lanzetta, P.A., Alvarez, L.J., Reinach, P.S., Candia, O.A., An improved assay for nanomole amounts of inorganic phosphate (1979) Anal Biochem, 100, pp. 95-97
  • Tummino, P.J., Gafni, A., Determination of the aggregation number of detergent micelles using steady-state fluorescence quenching (1993) Biophys J, 64, pp. 1580-1587
  • Seber, G.A.F., (1989) Nonlinear Regression, , C.J W, New York: John Wiley and Sons


---------- APA ----------
Dodes Traian, M.M., Cattoni, D.I., Levi, V. & González Flecha, F.L. (2012) . A two-stage model for lipid modulation of the activity of integral membrane proteins. PLoS ONE, 7(6).
---------- CHICAGO ----------
Dodes Traian, M.M., Cattoni, D.I., Levi, V., González Flecha, F.L. "A two-stage model for lipid modulation of the activity of integral membrane proteins" . PLoS ONE 7, no. 6 (2012).
---------- MLA ----------
Dodes Traian, M.M., Cattoni, D.I., Levi, V., González Flecha, F.L. "A two-stage model for lipid modulation of the activity of integral membrane proteins" . PLoS ONE, vol. 7, no. 6, 2012.
---------- VANCOUVER ----------
Dodes Traian, M.M., Cattoni, D.I., Levi, V., González Flecha, F.L. A two-stage model for lipid modulation of the activity of integral membrane proteins. PLoS ONE. 2012;7(6).