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

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Introduction Control of membrane remodeling is essential in clathrin-mediated endocytosis (CME) as different types and levels of curvature are required at each stage of the budding of clathrincoated vesicles [1,2]. Several of the proteins thought to play relevant roles in CME (dynamin, amphiphysin, endophilin and epsin) were recently shown to induce tubulation in protein-free spherical liposomes, indicating a potential role as mediators in the membrane remodeling observed during CME (3, 4, 5 6). The BAR (Bin, amphiphysin, Rvs) domain, found in amphiphysin, endophilins, and a wide variety of other proteins with or without known function in CME (7), is able to bind lipid membranes, generate tubulation (both in vivo and in vitro) and to sense bilayer curvature (5, 8). This versatile domain has a banana shape and dimerizes in membranes, giving rise to a positively charged concave surface that binds to lipid bilayers. This concave surface is likely to be the reason why the domain presents higher affinities for high curvature liposomes in vitro. Several BAR domains also present an N-terminal sequence that forms an amphipathic helix upon membrane binding (9). This sequence is here referred to as helix 0 (H0-NBAR). BAR domains presenting this sequence are called N-BAR and are able to bind to liposomes and induce tubulation with much higher efficiency, even tough sensitivity for curvature is lost (8). After a point mutation in H0-NBAR of a conserved hydrophobic residue (F) to an acidic residue (E), lipid binding and tubulation were abolished for endophilin (5), and reduced for the corresponding mutation in amphiphysin1 (8). Conservative mutations of the same residue (F to W) had no effect (5). These results point to an important role of H0-NBAR in membrane remodeling by N-BAR domains, and this role is likely to be dependent on membrane embedding of H0-NBAR. The exact function of H0-NBAR in membrane tubulation is however still elusive. The H0 fragment from BRAP (breast-cancer-associated protein)/Bin2, one of the first BAR domain containing proteins to be identified (10, 11), presents great homology to other N- terminal amphipathic fragments of BAR domain-containing proteins (Fig.1). The N-BAR domain of BRAP was already shown to tubulate liposomes in an identical fashion to other N- BAR domains. The mutations performed on the N-BAR domain from BRAP had also analogous effects in lipid tubulation as the corresponding mutants of the N-BAR domain of amphiphysin, corroborating that the same liposome tubulation mechanism was shared by the two proteins. Here we investigated the interaction of a peptide comprising the H0-NBAR fragment of BRAP with model lipid membranes. We performed a thorough study of the effects of partition of the N-BAR N-terminal domain to lipid membranes on both structure and dynamics of H0-NBAR itself and the interacting lipid membranes. We show that the N- terminal fragment of the N-BAR domain assumes in effect an alpha-helical structure upon membrane binding and that membrane binding is dependent on the presence of anionic phospholipids but virtually insensitive to both anionic lipid structure and liposome curvature. Through FRET (Förster resonance energy transfer) it is demonstrated that H0-NBAR dimerizes after incorporation in lipid membranes, providing a possible mechanism for generation of highorder oligomers of N-BAR domains. Monitoring the fluorescence of different membrane probes, membrane insertion of H0-NBAR is show to increase the packing of lipids both in the hydrophobic and headgroup regions of the bilayer. Finally, we also find that H0-NBAR is effective in inducing liposome fusion but has no liposome tubulation activity. Our results rule out the insertion of H0-NBAR in the exposed outer membrane leaflet as the mechanism of tubulation induced by N-BAR domains and point to a likely interplay between the membrane binding of H0-NBAR and the scaffold provided by the concave surface of the BAR domain. 3
Methods Materials Peptides H0-NBAR, H0-NBAR-EDANS(5-((2-aminoethyl)amino)naphthalene-1- sulfonic acid), H0-NBAR-FITC(fluorescein isothiocyanate), and H0-ENTH(Epsin N-terminal homology domain) were synthesized by Genemed Synthesis (San Francisco, CA). Labeling was achieved by conjugation on N-terminal end of the peptide. The purity was always higher than 95%. 1-Palmitoyl-2-oleoyl-sn-phosphocholine (POPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3- [Phospho-rac-(1-glycerol)] (POPG), 1-Palmitoyl-2-oleoyl-sn-glycero-3-(phospho-l-serine) (POPS), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4- yl) (NBD-DOPE), 1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-snglycero-phosphocholine (PC-NBD), 1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4- yl)amino]dodecanoyl]-sn-glycero-3-[phospho-rac-(1-glycerol)] (PG-NBD), 1-Oleoyl-2-[12- [(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-phosphoserine (PS-NBD), 1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-phosphate (PA-NBD), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Lissamine Rhodamine B Sulfonyl) (Rho-DOPE) were from Avanti Polar-Lipids (Alabaster, AL). 1,6-Diphenyl-1,3,5-hexatriene (DPH) and 5,6-carboxyfluorescein (5,6-CF) were from Molecular Probes (Eugene, OR). Brain extract from bovine brain (Folch fraction I) was from Sigma-Aldrich (St. Louis, MO). Fine chemicals were obtained from Merck (Darmstadt, Germany). All materials were used without further purification. Liposomes preparation The desired amount of phospholipids were mixed in chloroform and dried under a N 2(g) flow. The sample was then kept in vacuum overnight. Liposomes were prepared with buffer Hepes 20 mM, NaCl 150mM pH 7.4. The hydration step was performed with gentle addition of buffer followed by freeze-thaw cycles. Large unilamellar vesicles (LUV) were produced by extrusion through polycarbonate filters (12) in a Avestin extruder. Liposomes with 5,6-CF encapsulated were prepared by hydration with buffer Hepes 20 mM, NaCl 150mM pH 8.4 with 5mM 5,6-CF. After extrusion the suspension was passed through a 10ml Econo-Pac 10DG column Bio-Gel P-6DG gel with 6 kDa molecular weight exclusion) and eluted with buffer Hepes 20 mM, NaCl 150mM pH 8.4. CD spectroscopy CD spectroscopy was performed on a Jasco J-720 spectropolarimeter with a 450 W Xe lamp. Lipid suspensions were extruded using polycarbonate filters of 0.1 μm. Peptide concentration was 40 μM. Fluorescence Spectroscopy 4
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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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Page 123: BINDING ASSAYS OF INHIBITORS TOWARD
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Page 140 and 141: Introduction Quinolones are broad-s
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Page 146 and 147: placement of the protein around the
Page 148 and 149: ⎛ II t ⎛ R ⎞ 0 ρ () t = exp
Page 150 and 151: APPENDIX - Derivation of the FRET r
Page 152 and 153: 2 2w J ( t) = '2 R − R + w / 1
Page 154 and 155: [14] L. Plançon, M. Chami, L. Lete
Page 156 and 157: acceptors) (Eqs. 4-6) (⋅-⋅-⋅)
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Page 166 and 167: dynamin. Soon after, the same group
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Page 174 and 175: Steady-state fluorescence measureme
Page 176 and 177: Rho-DOPE (acceptor). The increase o
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Page 180 and 181: ound conformation of the BAR domain
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Page 184 and 185: Figure Legends Fig.1: N-terminal am
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Page 188 and 189: FIG. 3A FIG. 3B 19
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Page 192 and 193: FIG 6. 23
Page 194 and 195: FIG. 8 A B 25
Page 196 and 197: The signalling functions of PI(4,5)
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Page 202 and 203: zoxadiazol (NBD)-PC were obtained f
Page 204 and 205: Fig. 3. Fluorescence decays of diph
Page 206 and 207: CONCLUSIONS VII CONCLUSIONS Chapter
Page 208 and 209: CONCLUSIONS constants recovered by
Page 210 and 211: CONCLUSIONS Chapter IV ‐ Binding
Page 212 and 213: CONCLUSIONS Chapter VI - Clustering
Page 214 and 215: FINAL CONSIDERATIONS AND PROSPECTS
Page 216 and 217: BIBLIOGRAPHY IX BIBLIOGRAPHY Albert
Page 218 and 219: BIBLIOGRAPHY Phosphatidylinositol 4
Page 220 and 221: BIBLIOGRAPHY Glaubitz, C., Grobner,
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BIBLIOGRAPHY Koehorst, R. B. M., Sp
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BIBLIOGRAPHY Mishra, V. K., Palguna
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BIBLIOGRAPHY Pluschke, G., Hirota,
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BIBLIOGRAPHY Sperotto, M. M., and M
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BIBLIOGRAPHY Williamson, I. M., Alv
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