Home Life Sciences Interaction of the human N-Ras protein with lipid raft model membranes of varying degrees of complexity
Article
Licensed
Unlicensed Requires Authentication

Interaction of the human N-Ras protein with lipid raft model membranes of varying degrees of complexity

  • Alexander Vogel , Jörg Nikolaus , Katrin Weise , Gemma Triola , Herbert Waldmann , Roland Winter , Andreas Herrmann and Daniel Huster EMAIL logo
Published/Copyright: February 14, 2014
Biological Chemistry
From the journal Biological Chemistry Volume 395 Issue 7-8

Abstract

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance – PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol – using 2H solid-state nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

Keywords: 2H NMR; atomic force microscopy (AFM); confocal fluorescence microscopy lipid modification; membrane protein; order parameter
Corresponding author: Daniel Huster, Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstraße 16-18, D-04107 Leipzig, Germany; and Tata Institute of Fundamental Research, Department of Chemical Sciences, Homi Bhabha Road, Colaba, Mumbai 400 005, India, e-mail:
aPresent address: Yale University, 850 West Campus Dr., New Haven, CT 06516, USA.

Acknowledgments

The study was supported by the Deutsche Forschungsgemeinschaft [DFG HU 720/10-1 (D.H.), SFB 642 (R.W.), and SFB 740 (A.H.)]

References

Aittoniemi, J., Niemela, P.S., Hyvonen, M.T, Karttunen, M., and Vattulainen, I. (2007). Insight into the putative specific interactions between cholesterol, sphingomyelin and palmitoyl-oleoyl phosphatidylcholine. Biophys. J. 92, 1125–1137.10.1529/biophysj.106.088427Search in Google Scholar

Angelova, M., Soléau, S., Méléard, P., Faucon, F., and Bothorel, P. (1992). Preparation of giant vesicles by external AC electric fields. Kinetics and applications. In: Trends in Colloid and Interface Science VI. C. Helm, M. Lösche and H. Möhwald, eds. (Berlin/Heidelberg: Springer), pp. 127–131.Search in Google Scholar

Bader, B., Kuhn, K., Owen, D.J., Waldmann, H., Wittinghofer, A., and Kuhlmann, J. (2000). Bioorganic synthesis of lipid-modified proteins for the study of signal transduction. Nature 403, 223–226.10.1038/35003249Search in Google Scholar

Bartels, T., Lankalapalli, R.S., Bittman, R., Beyer, K., and Brown, M.F. (2008). Raftlike mixtures of sphingomyelin and cholesterol investigated by solid-state 2H NMR spectroscopy. J. Am. Chem. Soc. 130, 14521–14532.10.1021/ja801789tSearch in Google Scholar

Brown, D.A. and London, E. (2000). Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J. Biol. Chem. 275, 17221–17224.10.1074/jbc.R000005200Search in Google Scholar

Brunsveld, L., Kuhlmann, J., Alexandrov, K., Wittinghofer, A., Goody, R.S., and Waldmann, H.. (2006). Lipidated Ras and Rab peptides and proteins – synthesis, structure and function. Angew. Chem. Int. Ed. 45, 6622–6646.10.1002/anie.200600855Search in Google Scholar

Brunsveld, L., Waldmann, H. and Huster, D. (2009). Membrane binding of lipidated Ras peptides and proteins – the structural point of view. Biochim. Biophys. Acta 1788, 273–288.10.1016/j.bbamem.2008.08.006Search in Google Scholar

Bunge, A., Müller, P., Stöckl, M., Herrmann, A., and Huster, D. (2008). Characterization of the Ternary Mixture of Sphingomyelin, POPC, and cholesterol: support for an inhomogeneous lipid distribution at high temperatures. Biophys. J. 94, 2680–2690.10.1529/biophysj.107.112904Search in Google Scholar

Caroni, P. (2001). Actin cytoskeleton regulation through modulation of PI(4, 5)P-2 rafts. EMBO J. 20, 4332–4336.10.1093/emboj/20.16.4332Search in Google Scholar

Casey, P.J. (1995). Protein lipidation in cell signaling. Science 268, 221–225.10.1126/science.7716512Search in Google Scholar

de Almeida, R.F., M., Fedorov, A., and Prieto, M. (2003). Sphingomyelin/phosphatidylcholine/cholesterol phase diagram, boundaries and composition of lipid rafts. Biophys. J. 85, 2406–2416.10.1016/S0006-3495(03)74664-5Search in Google Scholar

Filippov, A., Oradd, G., and Lindblom, G. (2004). Lipid lateral diffusion in ordered and disordered phases in raft mixtures. Biophys. J. 86, 891–896.10.1016/S0006-3495(04)74164-8Search in Google Scholar

Gamier-Lhomme, M., Grelard, A., Byrne, R.D., Loudet, C., Dufourc, E.J., and Larijani, B. (2007). Probing the dynamics of intact cells and nuclear envelope precursor membrane vesicles by deuterium solid state NMR spectroscopy. Biochim. Biophys. Acta 1768, 2516–2527.10.1016/j.bbamem.2007.06.004Search in Google Scholar

Gorfe, A.A., Pellarin, R., and Caflisch, A. (2004). Membrane localization and flexibility of a lipidated ras peptide studied by molecular dynamics simulations. J. Am. Chem. Soc. 126, 15277–15286.10.1021/ja046607nSearch in Google Scholar

Gueldenhaupt, J., Rudack, T., Bachler, P., Mann, D., Triola, G., Waldmann, H., Koetting, C., and Gerwert, K. (2012). N-Ras forms dimers at POPC membranes. Biophys. J. 103, 1585–1593.10.1016/j.bpj.2012.08.043Search in Google Scholar

Haugh, J.M. and Lauffenburger, D.A. (1997). Physical modulation of intracellular signaling processes by locational regulation. Biophys. J. 72, 2014–2031.10.1016/S0006-3495(97)78846-5Search in Google Scholar

Huster, D., Vogel, A., Katzka, C., Scheidt, H.A., Binder, H., Dante, S., Gutberlet, T., Zschörnig, O., Waldmann, H., and Arnold, K. (2003). Membrane insertion of a lipidated Ras peptide studied by FTIR, solid-state NMR and neutron diffraction spectroscopy. J. Am. Chem. Soc. 125, 4070–4079.10.1021/ja0289245Search in Google Scholar

Ionova, I.V., Livshits, V.A., and Marsh, D. (2012). Phase diagram of ternary cholesterol/palmitoylsphingomyelin/palmitoyloleoyl-phosphatidylcholine mixtures, spin-label EPR study of lipid-raft formation. Biophys. J. 102, 1856–1865.10.1016/j.bpj.2012.03.043Search in Google Scholar

Ipsen, J.H., Karlstrom, G., Mouritsen, O.G., Wennerstrom, H., and Zuckermann, M.J. (1987). Phase-equilibria in the phosphatidylcholine-cholesterol system. Biochim. Biophys. Acta 905, 162–172.10.1016/0005-2736(87)90020-4Search in Google Scholar

Janosch, S., Nicolini, C., Ludolph, B., Peters, C., Volkert, M., Hazlet, T.L., Gratton, E., Waldmann, H., and Winter, R. (2004). Partitioning of dual-lipidated peptides into membrane microdomains, lipid sorting vs peptide aggregation. J. Am. Chem. Soc. 126, 7496–7503.10.1021/ja049922iSearch in Google Scholar PubMed

Janosi, L. and Gorfe, A.A. (2010). Segregation of negatively charged phospholipids by the polycationic and farnesylated membrane anchor of Kras. Biophys. J. 99, 3666–3674.10.1016/j.bpj.2010.10.031Search in Google Scholar PubMed PubMed Central

Janosi, L., Li, Z., Hancock, J.F., and Gorfe, A.A. (2012). Organization, dynamics and segregation of Ras nanoclusters in membrane domains. Proc. Natl. Acad. Sci. USA 109, 8097–8102.10.1073/pnas.1200773109Search in Google Scholar

Karnovsky, M.J., Kleinfeld, A.M., Hoover, R.L., and Klausner, R.D. (1982). The concept of lipid domains in membranes. J. Cell Biol. 94, 1–6.10.1083/jcb.94.1.1Search in Google Scholar

Kusumi, A., Subczynski, W.K., Pasenkiewiczgierula, M., Hyde, J.S., and Merkle, H. (1986). Spin-label studies on phosphatidylcholine-cholesterol membranes – effects of alkyl chain-length and unsaturation in the fluid phase. Biochim. Biophys. Acta 854, 307–317.10.1016/0005-2736(86)90124-0Search in Google Scholar

Kuzmin, P.I., Akimov, S.A., Chizmadzhev, Y.A., Zimmerberg, J., and Cohen, F.S. (2005). Line tension and interaction energies of membrane rafts calculated from lipid splay and tilt. Biophys. J. 88, 1120–1133.10.1529/biophysj.104.048223Search in Google Scholar PubMed PubMed Central

Leslie, M. (2011). Do lipid rafts exist? Science 334, 1046–1047.10.1126/science.334.6059.1046-bSearch in Google Scholar PubMed

Li, Z. and Gorfe, A.A. (2013). Deformation of a two-domain lipid bilayer due to asymmetric insertion of lipid-modified Ras paptides. Soft Matter 9, 11249–11256.10.1039/c3sm51388bSearch in Google Scholar

Li, Z., Janosi, L., and Gorfe, A.A. (2012). Formation and domain partitioning of H-ras peptide nanoclusters, effects of peptide concentration and lipid composition. J. Am. Chem. Soc. 134, 17278–17285.10.1021/ja307716zSearch in Google Scholar PubMed PubMed Central

Luzzati, V. and Husson, F. (1962). The structure of the liquid-crystalline phases of lipid-water systems. J. Cell Biol. 12, 207–219.10.1083/jcb.12.2.207Search in Google Scholar PubMed PubMed Central

Marsh, D. (2010). Liquid-ordered phases induced by cholesterol: A compendium of binary phase diagrams. Biochim. Biophys. Acta Biomembr. 1798, 688–699.10.1016/j.bbamem.2009.12.027Search in Google Scholar PubMed

McConnell, H.M. and Vrljic, M. (2003). Liquid-liquid immiscibility in membranes. Annu. Rev. Biophys. Biomol. Struct. 32, 469–492.10.1146/annurev.biophys.32.110601.141704Search in Google Scholar PubMed

McLaughlin, S., Wang, J.Y., Gambhir, A., and Murray, D. (2002). PIP2 and proteins, Interactions, organization and information flow. Annu. Rev. Biophys. Biomol. Struct. 31, 151–175.10.1146/annurev.biophys.31.082901.134259Search in Google Scholar

Murray, D., Ben-Tal, N., Honig, B., and McLaughlin, S. (1997). Electrostatic interaction of myristoylated proteins with membranes, simple physics, complicated biology. Structure 5, 985–989.10.1016/S0969-2126(97)00251-7Search in Google Scholar

Oradd, G., Westerman, P.W., and Lindblom, G. (2005). Lateral diffusion coefficients of separate lipid species in a ternary raft-forming bilayer, a Pfg-NMR multinuclear study. Biophys. J. 89, 315–320.10.1529/biophysj.105.061762Search in Google Scholar

Pagano, R.E., Cherry, R.J., and Chapman, D. (1973). Phase-transitions and heterogeneity in lipid bilayers. Science 181, 557–559.10.1126/science.181.4099.557Search in Google Scholar

Petit, V.A. and Edidin, M. (1974). Lateral phase separation of lipids in plasma-membranes – Effect of temperature on mobility of membrane antigens. Science 184, 1183–1185.10.1126/science.184.4142.1183Search in Google Scholar

Petrache, H.I., Dodd, S.W., and Brown, M.F. (2000). Area per lipid and acyl length distributions in fluid phosphatidylcholines determined by 2H NMR spectroscopy. Biophys. J. 79, 3172–3192.10.1016/S0006-3495(00)76551-9Search in Google Scholar

Peyker, A., Rocks, O., and Bastiaens, P.I.H. (2005). Imaging activation of two Ras isoforms simultaneously in a single cell. ChemBioChem 6, 78–85.10.1002/cbic.200400280Search in Google Scholar PubMed

Pike, L.J. (2006). Rafts defined, a report on the Keystone Symposium on Lipid Rafts and Cell Function. J. Lipid Res. 47, 1597–1598.10.1194/jlr.E600002-JLR200Search in Google Scholar PubMed

Polozov, I.V. and Gawrisch, K. (2006). Characterization of the liquid-ordered state by proton MAS NMR. Biophys. J. 90, 2051–2061.10.1529/biophysj.105.070441Search in Google Scholar PubMed PubMed Central

Prior, I.A., Muncke, C., Parton, R.G., and Hancock, J.F. (2003). Direct visualization of Ras proteins in spatially distinct cell surface microdomains. J. Cell Biol. 160, 165–170.10.1083/jcb.200209091Search in Google Scholar PubMed PubMed Central

Reinert, J.C. and Steim, J.M. (1970). Calorimetric detection of a membrane-lipid phase transition in living cells. Science 168, 1580–1582.10.1126/science.168.3939.1580Search in Google Scholar

Reuther, G., Tan, K.T., Kohler, J., Nowak, C., Pampel, A., Arnold, K., Kuhlmann, J., Waldmann, H., and Huster, D. (2006a). Structural model of the membrane-bound C terminus of lipid-modified human N-ras protein. Angew. Chem. Int. Ed. 45, 5387–5390.10.1002/anie.200504266Search in Google Scholar

Reuther, G., Tan, K.T., Vogel, A., Nowak, C., Arnold, K., Kuhlmann, J., Waldmann, H., and Huster, D. (2006b). The lipidated membrane anchor of full length N-Ras protein shows an extensive dynamics as revealed by solid-state NMR spectroscopy. J. Am. Chem. Soc. 128, 13840–13846.10.1021/ja063635sSearch in Google Scholar

Rocks, O., Peyker, A., Kahms, M., Verveer, P.J., Koerner, C., Lumbierres, M., Kuhlmann, J., Waldmann H., Wittinghofer, A., and Bastiaens, P.I.H. (2005). An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 307, 1746–1752.10.1126/science.1105654Search in Google Scholar

Scheidt, H., A., Meyer, T., Nikolaus, J., Baek, D.J. Haralampiev, I., Thomas, L., Bittman, R., Müller, P., Herrmann, A., and Huster, D. Cholesterol’s aliphatic side chain structure modulates membrane properties. (2013). Angew. Chem. Int. Ed. 52, 12848–12851.10.1002/anie.201306753Search in Google Scholar

Simons, K. and Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387, 569–572.10.1038/42408Search in Google Scholar

Simons, K. and Vanmeer, G. (1988). Lipid sorting in epithelial cells. Biochemistry 27, 6197–6202.10.1021/bi00417a001Search in Google Scholar

Singer, S.J. and Nicolson, G.L. (1972). Fluid mosaic model of structure of cell-membranes. Science 175, 720–731.10.1126/science.175.4023.720Search in Google Scholar

Stier, A. and Sackmann, E. (1973). Spin labels as enzyme substrates, Heterogeneous lipid distribution in liver microsomal membranes. Biochim. Biophys. Acta, Biomembr. 311, 400–408.10.1016/0005-2736(73)90320-9Search in Google Scholar

Veatch, S.L., Soubias, O., Keller, S.L., and Gawrisch, K. (2007a). Critical fluctuations in domain-forming lipid mixtures. Proc. Natl. Acad. Sci. USA 104, 17650–17655.10.1073/pnas.0703513104Search in Google Scholar PubMed PubMed Central

Veatch, S.L., Leung, S.S.W., Hancock, R.E.W., and Thewalt, J.L. (2007b). Fluorescent probes alter miscibility phase boundaries in ternary vesicles. J. Phys. Chem. B. 111, 502–504.10.1021/jp067636iSearch in Google Scholar PubMed

Viola, A., Schroeder, S., Sakakibara, Y., and Lanzavecchia, A. (1999). T lymphocyte costimulation mediated by reorganization of membrane microdomains. Science 283, 680–682.10.1126/science.283.5402.680Search in Google Scholar PubMed

Vogel, A., Katzka, C.P., Waldmann, H., Arnold, K., Brown, M.F., and Huster, D. (2005). Lipid modifications of a Ras peptide exhibit altered packing and mobility versus host membrane as detected by 2H solid-state NMR. J. Am. Chem. Soc. 127, 12263–12272.10.1021/ja051856cSearch in Google Scholar PubMed

Vogel, A., Tan, K.T., Waldmann, H., Feller, S.E., Brown, M.F., and Huster, D. (2007). Flexibility of ras lipid modifications studied by 2H solid-state NMR and molecular dynamics simulations. Biophys. J. 93, 2697–2712.10.1529/biophysj.107.104562Search in Google Scholar PubMed PubMed Central

Vogel, A., Reuther, G., Weise, K., Triola, G., Nikolaus, J., Tan, K.T., Nowak, C., Herrmann, A., Waldmann, H., Winter, R., et al. (2009). The lipid modifications of Ras that sense membrane environments and induce local enrichment. Angew. Chem. Int. Ed. 48, 8784–8787.10.1002/anie.200903396Search in Google Scholar PubMed

Vogel, A., Reuther, G., Roark, M.B., Tan, K.T., Waldmann, H., Feller, S.E., and Huster, D. (2010). Backbone conformational flexibility of the lipid modified membrane anchor of the human N-Ras protein investigated by solid-state NMR and molecular dynamics simulation. Biochim. Biophys. Acta 1798, 275–285.10.1016/j.bbamem.2009.09.023Search in Google Scholar PubMed

Volkert, M., Uwai, K., Tebbe, A., Popkirova, B., Wagner, M., Kuhlmann, J., and Waldmann, H. (2003). Synthesis and biological activity of photoactivatable N-Ras peptides and proteins. J. Am. Chem. Soc. 125, 12749–12758.10.1021/ja036178dSearch in Google Scholar PubMed

Wang, T.Y., Leventis, R., and Silvius, J.R. (2001). Partitioning of lipidated peptide sequences into liquid-ordered lipid domains in model and biological membranes. Biochemistry 40, 13031–13040.10.1021/bi0112311Search in Google Scholar PubMed

Weise, K., Triola, G., Brunsveld, L., Waldmann, H., and Winter, R. (2009). Influence of the lipidation motif on the partitioning and association of N-Ras in model membrane subdomains. J. Am. Chem. Soc. 131, 1557–1564.10.1021/ja808691rSearch in Google Scholar PubMed

Weise, K., Kapoor, S., Denter, C., Nikolaus, J., Opitz, N., Koch, S., Triola, G., Herrmann, A., Waldmann H., and Winter, R. (2011). Membrane-mediated induction and sorting of K-Ras microdomain signaling platforms. J. Am. Chem. Soc. 133, 880–887.10.1021/ja107532qSearch in Google Scholar PubMed

Wittinghofer, A. and Waldmann H. (2000). Ras – A molecular switch involved in tumor formation. Angew. Chem. Int. Ed. 39, 4192–4214.10.1002/1521-3773(20001201)39:23<4192::AID-ANIE4192>3.0.CO;2-YSearch in Google Scholar

Zacharias, D.A., Violin, J.D., Newton, A.C., and Tsien, R.Y. (2002). Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916.10.1126/science.1068539Search in Google Scholar

Received: 2013-12-12
Accepted: 2014-2-6
Published Online: 2014-2-14
Published in Print: 2014-7-1

©2014 by Walter de Gruyter Berlin/Boston

Articles in the same Issue

  1. Frontmatter
  2. Guest Editorial
  3. Highlight: conformational transitions in macromolecular interactions
  4. Single-molecule spectroscopy of unfolded proteins and chaperonin action
  5. Influence of the polypeptide environment next to amyloidogenic peptides on fibril formation
  6. Structure of large dsDNA viruses
  7. Functional aspects of extracellular cyclophilins
  8. Generic tools for conditionally altering protein abundance and phenotypes on demand
  9. Structural insights into calmodulin/Munc13 interaction
  10. Interaction of linear polyamines with negatively charged phospholipids: the effect of polyamine charge distance
  11. Interaction of the human N-Ras protein with lipid raft model membranes of varying degrees of complexity
  12. Lanthanides as substitutes for calcium ions in the activation of plant α-type phospholipase D
  13. Insights from reconstitution reactions of COPII vesicle formation using pure components and low mechanical perturbation
  14. Identification of key residues in the formate channel FocA that control import and export of formate
  15. Twin-arginine translocation-arresting protein regions contact TatA and TatB
  16. Biophysical and biochemical analysis of hnRNP K: arginine methylation, reversible aggregation and combinatorial binding to nucleic acids
  17. An ancient oxidoreductase making differential use of its cofactors
  18. Biophysical characterization of polyomavirus minor capsid proteins
  19. Structural basis for PTPA interaction with the invariant C-terminal tail of PP2A
  20. Correlating structure and ligand affinity in drug discovery: a cautionary tale involving second shell residues
  21. Thermodynamic signatures in macromolecular interactions involving conformational flexibility
https://doi.org/10.1515/hsz-2013-0294
Keywords for this article
2H NMR; atomic force microscopy (AFM); confocal fluorescence microscopy lipid modification; membrane protein; order parameter

Articles in the same Issue

  1. Frontmatter
  2. Guest Editorial
  3. Highlight: conformational transitions in macromolecular interactions
  4. Single-molecule spectroscopy of unfolded proteins and chaperonin action
  5. Influence of the polypeptide environment next to amyloidogenic peptides on fibril formation
  6. Structure of large dsDNA viruses
  7. Functional aspects of extracellular cyclophilins
  8. Generic tools for conditionally altering protein abundance and phenotypes on demand
  9. Structural insights into calmodulin/Munc13 interaction
  10. Interaction of linear polyamines with negatively charged phospholipids: the effect of polyamine charge distance
  11. Interaction of the human N-Ras protein with lipid raft model membranes of varying degrees of complexity
  12. Lanthanides as substitutes for calcium ions in the activation of plant α-type phospholipase D
  13. Insights from reconstitution reactions of COPII vesicle formation using pure components and low mechanical perturbation
  14. Identification of key residues in the formate channel FocA that control import and export of formate
  15. Twin-arginine translocation-arresting protein regions contact TatA and TatB
  16. Biophysical and biochemical analysis of hnRNP K: arginine methylation, reversible aggregation and combinatorial binding to nucleic acids
  17. An ancient oxidoreductase making differential use of its cofactors
  18. Biophysical characterization of polyomavirus minor capsid proteins
  19. Structural basis for PTPA interaction with the invariant C-terminal tail of PP2A
  20. Correlating structure and ligand affinity in drug discovery: a cautionary tale involving second shell residues
  21. Thermodynamic signatures in macromolecular interactions involving conformational flexibility
Sign up now to receive a 20% welcome discount
Institutional Access
How does access work?
Have an idea on how to improve our website?
Please write us.
© 2025 De Gruyter Brill
Downloaded on 5.12.2025 from https://www.degruyterbrill.com/document/doi/10.1515/hsz-2013-0294/html
Scroll to top button