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

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Figure Legends Fig.1: N-terminal amphipatic helix of the BAR domain of BRAP. A – Alignments of N- terminal sequences (H0) of BRAP/Bin2 and several amphiphysins (Amph) show great degree of homology (h-Human, d-Drosophila, r-Rat). B – Helical wheel representation of H0 from BRAP. Numbers indicate position of the aminoacid. Hydrophobic residues are show in grey. Fig. 2: The N-terminal sequence of the BAR domain is alpha-helical in anionic liposomes. CD spectrum of H0 in buffer (---), in the presence of POPC (●) and POPG liposomes (―). Lipid concentration was 1mM. Fig. 3: Partition of H0 to bilayers is only sensitive to charge. A - Increase of fluorescence emission anisotropy of H0-EDANS with lipid concentration. H0-EDANS in the presence of POPC (▲), POPG (○) and POPS (■) liposomes of 100 nm diameter. INSET: Efficiencies of energy transfer from H0-NBAR-EDANS to NBD-labeled phospholipids (PX-NBD) in POPG liposomes. Bars show the difference in FRET efficiency (E) relative to the value obtained for PG-NBD (E PG- NBD = 0.38). Concentration of PX-NBD in POPG bilayers was 2% (mol/mol). B - Increase of fluorescence emission anisotropy of H0-EDANS with POPG concentration for different liposome sizes. H0-EDANS in the presence of POPG liposomes with 30 (∆), 100 (○) and 400 (■) nm radius. Fluorescence emission anisotropies were measured with excitation wavelength of 340 nm and emission wavelength of 460 nm. In both panels, the lines are the fits of Equation 1 to the data, as described in the text. Fig. 4: H0-NBAR promotes liposome fusion in both POPC and POPG. Decrease in fluorescence emission intensities of NBD-DOPE 15 min. after mixing of liposomes with 2% NBD- DOPE and liposomes with 2% Rho-DOPE in the presence and in the absence of H0-NBAR. Results obtained with H0-ENTH are shown for comparison. Fluorescence intensity in the absence of acceptor was measured and no significant donor fluorescence bleaching was detected. Fluorescence intensities were obtained with excitation and emission wavelengths set to 460 and 510 nm respectively. Fig. 5: H0 forms an antiparallel dimer in POPG bilayers. A- FRET efficiencies determined from the integration of H0-EDANS fluorescence decays in the presence of increasing fraction of acceptors (H0-FITC) (■) at a L/P = 1000 and 2000. Simulations for FRET between monomers (···), parallel dimers (---) or trimers (-⋅-) do not describe the data accurately. The simulation which provided a more accurate description of the data was obtained for an antiparallel dimer (⎯) in which EDANS and FITC are separated by 43 Å (the estimated size of H0-NBAR is 49.5 Å). Insensitivity to L/P ratios further supports the derived formalisms. B – Dependence of the longer rotational correlation time (φ 2 ) of the fluorescence anisotropy decay of H0-NBAR-FITC on the concentration of peptide in buffer. The dotted line is the expected φ 2 for a H0-NBAR monomer. The dynamics of H0-NBAR-FITC is virtually unaffected by the increase in concentration and the recovered longer correlation time is in agreement with the rotation of a H0 monomer (see text). All samples contained the same concentration of H0-NBAR-FITC, and different concentrations of H0- NBAR were obtained through addition of unlabelled peptide. Fig. 6: Translocation of H0-BAR in POPG liposomes is very slow. Stern-Volmer plots for fluorescence emission quenching of H0-NBAR-EDANS by iodide. H0-EDANS in buffer (○), in the presence of POPG liposomes with a 3 min. (●) and 40 min. (▲) incubation time prior to addition of iodide quencher. Permeation of iodide across the bilayer is slow (see Inset) and after 40 min. the amount of H0-NBAR-EDANS that translocated through the bilayer is estimated to be a maximum of 4%. INSET: Permeation of I - across POPG liposomes as measured by fluorescence quenching of encapsulated 5,6-CF. 15
Fig. 7: H0-NBAR increases phospholipid packing. A – Effect of unlabeled H0-NBAR on the fluorescence emission anisotropy of NBD-DOPE (•). NBD-DOPE fluorescence is sensitive to changes in the headgroup region of the bilayer. B - Effect of unlabelled H0-NBAR on the fluorescence emission anisotropy of DPH (•). DPH fluorescence is sensitive to changes in the acylchain region of the bilayer. For comparison, the effect on both probes of an amphipathic peptide which is not expected to insert in the bilayer (H0-ENTH) is also presented (○). Lines are mere guides to the eye. Fig. 8: H0-NBAR promotes liposome fusion but does not alter liposome morphology. A – TEM micrograph of POPG liposomes. B – TEM micrograph of POPG liposomes after incubation with H0-NBAR (L/P = 10). 16
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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 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 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
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Page 180 and 181: ound conformation of the BAR domain
Page 182 and 183: 19. Fernandes, F., L. M. S. Loura,
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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,
Page 228 and 229: BIBLIOGRAPHY Sperotto, M. M., and M
Page 230: BIBLIOGRAPHY Williamson, I. M., Alv
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