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

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where η is the viscosity of the medium, V is the molar volume of the molecule, R is the ideal gas constant and T is the temperature. From Equation 8 and taking into consideration the hydration volume for a protein (22) the expected φ 2 of monomeric H0-NBAR is 1.54 ns (Fig. 5B). Hence, H0-NBAR dynamics in solution is as expected for a monomer and is independent of concentration up to 20 μM of peptide. H0-NBAR does not translocate efficiently across the bilayer When bound to the full N-BAR domain, H0-NBAR is expected to interact with only one monolayer. The structural changes induced in the exposed monolayer and the resulting monolayer asymmetry could be responsible for membrane bending mediated by N-BAR, in a similar mechanism as the one observed for the ENTH domain of epsin after PIP(4,5) 2 binding (23). In order to study if the H0-NBAR is able to induce by itself the sort of membrane bending observed with full N-BAR domains it is necessary to determine if the peptide, after interaction with anionic liposomes, remains bound to the outer monolayer, or if it exhibits fast translocation across the bilayer. If H0-NBAR translocated efficiently across the bilayer, effects on both monolayers would limit monolayer asymmetry and consequently H0-NBAR would not behave in a comparable manner as the helix 0 in the N-terminal segment of N-BAR. To evaluate this, the following methodology was applied. The iodide ion exhibits low permeability across lipid bilayers (Fig. 6- Inset), and by measuring the degree of fluorescence quenching induced by I - on H0-NBAR-EDANS it is possible to estimate the fraction of H0- NBAR-EDANS that translocated across the bilayer. Two sets of samples were measured. In the first, 1mM POPG liposomes were incubated with 2μM of H0-NBAR-EDANS for only 3 minutes, as this amount of time guarantees almost complete binding of the peptide to the membrane (results not shown). In the second set of samples, the same concentrations of POPG liposomes and H0-NBAR-EDANS were incubated for 40 min. The fluorescence intensities were measured immediately before and after addition of KI and the Stern-Volmer plots for fluorescence quenching were obtained (Fig. 6). It is clear that the difference between the degree of exposure of H0-NBAR to KI is minimal between the two data sets. Assuming that H0-NBAR-EDANS in the inside of vesicles was 100% inaccessible to KI, after 40 minutes of incubation, there was only a maximum of 4 % translocated H0-NBAR. H0-NBAR increases packing of anionic phospholipids at a higher degree than a control peptide The effect of unlabeled H0-NBAR in the packing and dynamics of phospholipids was measured through the fluorescence anisotropy of two lipid membrane probes, NBD-DOPE and DPH. The first is a good probe for the headgroup region of the bilayer while the second is a well known probe of acyl chain packing. The effects of adding increasing amounts of H0- NBAR to POPG liposomes loaded with 1 % (mol/mol) of fluorescent probe are shown in Figure 7. The results obtained with H0-ENTH are also shown for comparison. ENTH is thought to be able to tubulate liposomes in the presence of PIP(4,5) 2 through insertion of H0- ENTH in the acyl-chain. In the absence of this lipid, H0-ENTH is unable to penetrate the membrane (6). It should be stressed that the induced probe anisotropy increase is not related to a decrease of its lifetime (Perrin equation, [14]), since the probe fluorescence intensity is invariant (there is no fluorescence quenching), upon peptide addition (results not shown). H0-NBAR clearly rigidifies both the acyl-chains and the headgroup region of the bilayer. H0-ENTH has only minor effects on the fluorescence anisotropy of both probes even 9
at very high concentrations, confirming that H0-NBAR is particularly rigidifying for phospholipid packing. H0-NBAR is not able to tubulate lipid membranes in the absence of the scaffold domain of N-BAR. TEM was used to monitor the effects of H0-NBAR on liposome morphology (Fig. 8). H0-NBAR is clearly not able to induce tubulation of liposomes composed of pure synthetic phospholipids (POPG) (Fig. 8) or of lipid brain extracts (results not shown). The only noticeable effect of H0-NBAR was the fusion of the liposomes, without a significant change in their morphology or size. Different P/L ratios produced similar results. Discussion Proteins are believed to be able to induce membrane bending by means of three mechanisms, namely the scaffold, local spontaneous curvature and the bilayer-couple mechanism (24). In the scaffold mechanism, proteins present and force their intrinsic curvature to the lipid membrane. This intrinsic curvature can be the result of tertiary structure or of the surface from a protein network. In the local spontaneous curvature hypothesis, a shallow insertion in the lipid membrane of an amphipathic moiety from a protein induces local perturbation of the packing of lipid headgroups resulting in higher local curvature. Finally in the bilayer-couple mechanism, the insertion of an amphipathic helix in the lipid bilayer could result in an increase of the area of the monolayer where the protein is inserted that is compensated by an increase of bilayer curvature. The presence of H0-NBAR greatly enhances the liposome tubulation activity of BAR domains. Several theories have been recently presented to explain the relevance of H0-NBAR in this phenomenon. It is feasible that the sole function of H0-NBAR is the increase in residence time of the BAR domain in the membrane by tight binding to the hydrophobic interior of the bilayer, but it is also possible that this segment is important in the oligomerization of BAR domains in the membrane, since cross-linking experiments suggest that BAR domains exist in membranes as high-order oligomers [5, 25]. Therefore, N-BAR domains could induce remodeling of membranes by the scaffold, the local spontaneous curvature and the bilayer-couple mechanisms or by a combination of them. The concave shape of the BAR dimer can act as a scaffold, forcing the membrane curvature to adapt to its own curvature. Insertion of the amphipathic helix could also act through either the bilayer-couple or the local spontaneous curvature mechanism. Here we showed that insertion of H0-NBAR in the lipid bilayer is unable to induce significant changes in lipid membrane morphology. Recently, some authors reported that highly amphipathic peptides induced liposomes or supported lipid bilayers to adopt tubular structures when present at very high concentrations (L/P
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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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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) (⋅-⋅-⋅)
Page 158 and 159: FIGURE 1A FIGURE 1B 130
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Page 166 and 167: dynamin. Soon after, the same group
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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 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 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,
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,
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BIBLIOGRAPHY Sperotto, M. M., and M
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BIBLIOGRAPHY Williamson, I. M., Alv
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