Biophysical studies of membrane proteins/peptides. Interaction with ...

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ound conformation of the BAR domain through strong hydrophobic interactions with the membrane. Although partition of H0-NBAR to anionic liposomes is very efficient, it is unlikely to be higher than the partition of the BAR domain itself, as the concave surface of the BAR domain dimer is already strongly positively charged. Partition of H0-NBAR to anionic bilayers disturbs both the headgroup as the acyl-chain regions of the bilayer. H0-NBAR is more disturbing to the bilayer than H0-ENTH, and this can be the result of H0-NBAR dimerization in the membrane environment, as dimerization of amphipathic peptides was previously shown to be particularly disturbing to bilayer structure (28). The dimerization of H0-NBAR explains the detection of high-order oligomers of N-BAR domains (5,25), and if effective in the full BAR domain, can be the mechanism by which H0-NBAR provides a more favorable framework for tubulation mediated by N-BAR (7). A recent molecular dynamics study (29) showed binding of N-BAR domains to a lipid membrane resulting in generation of membrane curvature through the scaffold mechanism. Results suggested that the main role of the N-terminal amphipathic helix of N-BAR was to favor the orientation of the N-BAR domain that allowed direct interaction between the membrane and the protein concave face. In effect, from our result, it is clear that the membrane bending activity of the N-BAR domain must be achieved through the scaffold mechanism in which the BAR domain presents its concave surface to the membrane, and forces the membrane to adopt the same curvature, while H0-NBAR only plays a promoting role in this process, either by: i) enhancing the membrane affinity of the full N-BAR domain; ii) mediating N-BAR high order oligomerization and stimulating the increase of local concentrations of the protein; iii)increasing lipid packing and facilitating curvature generation; iv) forcing the protein to present its concave face to the membrane or by a combination of these mechanisms. Acknowledgments F. F. acknowledges grant SFRH/BD/14282/2003 from FCT (Portugal). A.F. acknowledges grant SFRH/BPD/26150/2005 from FCT (Portugal) This work was funded by FCT (Portugal) under the program POCI. 11
References 1. Farsad, K., and P. De Camilli. 2003. Mechanisms of membrane deformation. Curr. Opin. Cell Biol. 15:372-381. 2. McMahon, H. T., and J. L. Gallop. 2005. Membrane curvature and mechanisms of dynamic cell membrane remodeling. Nature 438:590-596. 3. Takei, K., V. Haucke, V. Slepnev, K. Farsad, M. Salazar, H. Chen, and P. De Camilli. 1998. Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes. Cell 94:131-141. 4. Takei, K., V. I. Slepnev, V. Haucke, P. De Camilli. 1999. Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. Nat. Cell Biol. 1:33- 39. 5. Farsad, K., N. Ringstad, K. Takei, S. R. Floyd, K. Rose, and P. De Camilli. 2001. Generation of high curvature membranes mediated by direct endophilin bilayer interactions. J. Cell Biol. 155:193-200. 6. Ford, M. G. J., I. G. Mills, B. J. Peter, Y. Vallis, G. J. K. Praefcke, P. R. Evans, and H. T. McMahon, 2002. Curvature of clathrin-coated pits driven by epsin. Nature 419:361- 366. 7. Gallop, J. L., and H. T. McMahon. 2005. BAR domains and membrane curvature: bringing your curves to the BAR. Biochem. Soc. Symp. 72:223-31. 8. Peter, B. J., H. M. Kent, I.G. Mills, Y. Vallis, P. J. G. Butler, P. R. Evans, and H. T. McMahon. 2004. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303:495-499. 9. Gallop, J. L., C. C. Jao, H. M. Kent, P. J. G. Butler, P. R. Evans, R. Langen, and H. T. McMahon. 2006. Mechanism of endophilin N-BAR domain-mediated membrane curvature. EMBO J. 25:2898-2910. 10. Ge, K., and G. C. Prendergast. 2000. Bin2, a functionally nonredundant member of the BAR adaptor gene family. Genomics 67: 210-220. 11. Greiner, J., M. Ringhoffer, M. Taniguchi, T. Hauser, A. Schmitt, H. Döhner, and M. Schmitt. 2003. Characterization of several leukemia-associated antigens inducing humoral immune responses in acute and chronic myeloid leukemia. Int. J. Cancer 106:224-231. 12. Mayer, L.D., M. J. Hope, and P. R. Cullis. 1986. Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim. Biophys. Acta 858:161-168. 13. Loura, L. M. S., A. Fedorov, and M. Prieto. 2001. Fluid-fluid membrane microheterogeneity: a fluorescence resonance energy transfer study. Biophys. J. 80: 776- 788. 14. Lakowicz J.R. 1999. Principles of fluorescence spectroscopy. Kluwer Academic/Plenum Publishers, NY. 15. Loura, L.M.S., A. Fedorov, and M. Prieto. 1996. Resonance energy transfer in a model system of membranes: application to gel and liquid crystalline phases. Biophys. J. 71:1823-1836. 16. de Almeida, R. F. M. , L. S. M. Loura, M. Prieto, A. Watts, A. Fedorov, and F. J. Barrantes. 2006. Structure and dynamics of the γ-M4 transmembrane domain of the acetylcholine receptor in lipid bilayers: insights into receptor assembly and function Mol. Membr. Biol. 23: 305-315. 17. Hudson, E. N., and G. Weber. 1973. Synthesis and characterization of two fluorescent sulfhydryl reagents. Biochemistry 12:4154-4161. 18. Santos, N. C., M. Prieto, and M. A. R. B. Castanho. 2003. Quantifying molecular partition into model systems of biomembranes: an emphasis on optical spectroscopic methods. Biochimica et Biophysica Acta 1612: 123-135. 12
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UNIVERSIDADE TÉCNICA DE LISBOA INS
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Fernandes, F., Loura, L. M. S., Fed
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Ao Pedro e Hugo, amigos de longa da
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2.2. - Peptides as models 2.3. - Am
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ABBREVIATIONS AND SYMBOL LIST ABBRE
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RESUMO RESUMO As biomembranas são
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SINOPSE SINOPSE Nas últimas duas d
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SINOPSE podem fornecer informação
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SINOPSE aceitantes. Dadores mais pr
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OUTLINE OUTLINE The last two decade
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OUTLINE membranes with a distributi
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OUTLINE BAR domains (tubulation of
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ion diffusion, as the energy requir
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acid. If no more groups are linked
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sphingomyelin, the most abundant sp
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signal for neighbouring cells to ph
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functional role in process such as
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in the L α phase, while lateral di
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1.7. Lateral heterogeneity in lipid
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the vesicle through bilayer deforma
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Figure I.9 - Depiction of the sever
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Figure I.11 - Experimentally obtain
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drying into a film and ressuspensio
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deactivating agents. Zwitterionic d
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amphipatic helices (see Section 2.3
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size corresponding to the hydrophob
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changes abruptly in the interfacial
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thickness of the bilayer (Section 1
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some α-helical membrane proteins a
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The formation of a lipid population
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homogeneous distribution of lipids
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Figure I.20 - Relative binding cons
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2.9. Lipid phase preferential parti
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In Figure I.21, theoretical simulat
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PROTEIN-PROTEIN AND PROTEIN-LIPID I
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2430 Biophysical Journal Volume 85
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2432 Fernandes et al. Coat protein
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2434 Fernandes et al. leads to an a
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2436 Fernandes et al. FIGURE 4 (A)
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2438 Fernandes et al. section of th
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2440 Fernandes et al. tein oligomer
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QUANTIFICATION OF PROTEIN-LIPID SEL
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FRET Study of Protein-Lipid Selecti
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BINDING OF INHIBITORS TO A PUTATIVE
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BINDING OF INHIBITORS TO A PUTATIVE
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INTERACTION OF THE INDOLE CLASS OF
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5272 Biochemistry, Vol. 45, No. 16,
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BINDING ASSAYS OF INHIBITORS TOWARD
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1778 F. Fernandes et al. / Biochimi
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1780 F. Fernandes et al. / Biochimi
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Page 140 and 141: Introduction Quinolones are broad-s
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Page 150 and 151: APPENDIX - Derivation of the FRET r
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Page 206 and 207: CONCLUSIONS VII CONCLUSIONS Chapter
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Page 216 and 217: BIBLIOGRAPHY IX BIBLIOGRAPHY Albert
Page 218 and 219: BIBLIOGRAPHY Phosphatidylinositol 4
Page 220 and 221: BIBLIOGRAPHY Glaubitz, C., Grobner,
Page 222 and 223: BIBLIOGRAPHY Koehorst, R. B. M., Sp
Page 224 and 225: BIBLIOGRAPHY Mishra, V. K., Palguna
Page 226 and 227: BIBLIOGRAPHY Pluschke, G., Hirota,
Page 228 and 229: BIBLIOGRAPHY Sperotto, M. M., and M
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BIBLIOGRAPHY Williamson, I. M., Alv
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