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

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dynamin. Soon after, the same group observed a similar behaviour with another protein, endophilin, that was also know to bind dynamin (Farsad et al., 2001). Amphiphysin and endophilin both share a homologous N-terminal amino acid stretch which was shown to be crucial for liposome tubulation activity (Farsad et al., 2001). This domain is highly conserved in both proteins and is found in the N-terminal of other proteins. Due to its presence in the bin, amphiphysin and Rvs161/167 proteins (all amphiphysin family members), it was called BAR domain (Ge and Prendergast, 2000). BAR domains are responsible for the dimerization of amphiphysin in lipid membranes and are able to induce tubulation of liposomes isolated from the rest of the protein (Farsad et al., 2001). The structure of the BAR domain of amphiphysin was solved by McMahon and co-workers (Peter et al., 2004). It is a crescent-shaped dimer, and each monomer has three kinked α-helices (Figure V-1). Figure V-1: Crystal structure of the BAR domain of amphiphysin from Drosophila (taken from Peter et al., 2004). McMahon and co-workers also observed increased amphiphysin binding efficiency to liposomes of higher curvature, and suggested that this was due to a better fitting of the concave surface of the BAR domain dimer to the liposome surface. When a short N-terminal stretch predicted to be an amphipatic helix (Helix-0 or H0) was removed, liposome binding efficiencies decreased. Tubulation, however, was still detected, albeit much less efficient. This N-terminal stretch is not present in all BAR domains, and BAR domains containing it are called N-BAR domains (Peter et al., 2004; Gallop and McMahon, 2005). The authors also suggested that the function of this N- terminal segment was to insert into the bilayer as an amphipatic helix. This insertion would lead to an asymmetry between the inner and outer leaflet causing an increase of positive curvature, whereas the concave surface of the BAR domain acts as a scaffold to the membrane curvature (Gallop et al., 2006; Zimmerberg and Kozlov, 2006). In fact, the rigidity of the crescent dimer shape is crucial for liposome tubulation (Masuda et al., 138
INTERACTION OF HELIX-0 OF THE N-BAR DOMAIN WITH LIPID BILAYERS 2006). The N-terminal amphipatic segments were also proposed to act as mediators of oligomerization between BAR domain dimers, increasing the efficiency of the tubulation process (Richnau et al., 2004; Gallop and McMahon, 2005) A model for the function of amphiphysin and other BAR proteins involved in clathrin mediated endocytosis is represented in Figure V-2. Amphiphysin can act in clathrin mediated endocytosis by binding to the nascent vesicle on the early stages of invagination, either through the BAR domain itself or through interaction with other proteins. Following this, the BAR domain can contribute to the generation of the bud neck with the required dimensions, and as the pit matures, more amphiphysin is recruited to the vesicle neck due to its curvature sensing properties. Dynamin is assembled together with amphiphysin and GTP hydrolysis leads to vesicle fission (Yoshida et al., 2004). Figure V-2: Possible mechanism of action of amphiphysin in clathrin mediated endocytosis. Coat components (clathrin) were omitted (taken from Yoshida et al., 2004). 139
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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 136 and 137: apparently the most significant as
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Page 140 and 141: Introduction Quinolones are broad-s
Page 142 and 143: [9-12]. Having this into considerat
Page 144 and 145: any case, very similar, probably wi
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) (⋅-⋅-⋅)
Page 158 and 159: FIGURE 1A FIGURE 1B 130
Page 164 and 165: 136
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Page 172 and 173: Introduction Control of membrane re
Page 174 and 175: Steady-state fluorescence measureme
Page 176 and 177: Rho-DOPE (acceptor). The increase o
Page 178 and 179: where η is the viscosity of the me
Page 180 and 181: ound conformation of the BAR domain
Page 182 and 183: 19. Fernandes, F., L. M. S. Loura,
Page 184 and 185: Figure Legends Fig.1: N-terminal am
Page 186 and 187: FIG. 1A FIG.1B 17
Page 188 and 189: FIG. 3A FIG. 3B 19
Page 190 and 191: FIG.5A 21
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 200 and 201: 174
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
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BIBLIOGRAPHY IX BIBLIOGRAPHY Albert
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BIBLIOGRAPHY Phosphatidylinositol 4
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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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