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

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[9-12]. Having this into consideration, OmpF proteoliposomes were prepared by direct incorporation into preformed DMPC liposomes, by a well established methodology [10, 13-15]. Briefly, an adequate volume (~ 2.6 ml) of DMPC liposome suspension (~ 2.0 mM) in Hepes buffer, prepared according usual procedures [16], is added to OmpF (0.45 mg) in a Hepes buffer solution with 0.4% of oPOE. The lipid/protein mole ratio is always near 1000 and the total volume of the mixture assures a final concentration of oPOE lower than the value of its CMC (0.23%). After an efficient homogenization by gentle stirring, the mixture is incubated 15 min at room temperature followed by 1 h on ice. The detergent is then adsorbed onto SM2 Bio-Beads ® from Bio-Rad (Hercules, CA) at a concentration of 0.2 g of Bio-Beads/ml, by gently shaking of the suspension during a period of 3 h. After this time, a second portion of the same amount of Bio-Beads is added and the suspension is again shaken for another 3 h. In the end of this period, proteoliposomes are gently removed by decanting the Bio-Beads. The orientation of OmpF by this reconstitution procedure is considered similar to that observed in the bacterial membranes, based in experimental and molecular dynamics studies [17,18]. As the study to be performed is based on the fluorescence of OmpF, the presence of protein not inserted in the membrane would lead to errors in the final results. The suspension of proteoliposomes was therefore submitted to an ultracentrifugation (80000 g, 4 ºC, 2 h) in order to remove any traces of protein not incorporated. After this procedure the supernatant was rejected and the pellet suspended in Hepes buffer. Protein was quantified in the liposomes and in the supernatant. The percentage of incorporation by this methodology was always higher than 76%. In the final step of the procedure the proteoliposomes are sequentially extruded through 200 and 100 nm polycarbonate membranes from Nucleopore (Kent, WA). Quenching of OmpF fluorescence by CP The fluorescence quenching studies were achieved by successive additions of a constant volume (10 µl) of CP solution (~ 296 µM) to the cuvette (final concentration range: 0- 38µM) containing a constant amount of OmpF (~ 0.45 µM) incorporated in the liposomes. Fluorescence spectra were measured with an excitation wavelength of 290 nm. Inner filter effects and dilution of the solution were taken into account. 114
BINDING OF A QUINOLONE ANTIBIOTIC TO BACTERIAL PORIN OmpF Theoretical Modelling CP quenches OmpF fluorescence through a Förster resonance energy transfer (FRET) mechanism. FRET efficiencies (E) are calculated from the extent of fluorescence emission quenching of the donor induced by the presence of acceptors, IDA E = 1− = 1− I D ∞ ∫ DA 0 ∞ 0 i ∫ i D () t dt () t dt where I DA and I D are the steady-state fluorescence intensities of the donor in the presence and absence of acceptors respectively. i DA (t) and i D (t) are the donor fluorescence decays in the presence and absence of acceptors respectively. Although there is a contribution of static quenching to FRET in OmpF-CP complexes, Eq.(1) still holds since this is taken into account as described below (Eqs. 8-12). As seen in Fig.3, CP absorbance overlaps with the OmpF’s fluorescence emission spectrum (from both tryptophans 214 and 61), and the overlap integral (J) is calculated as: (1) ∫ ( ) 4 J = f λ ⋅ε(λ) ⋅λ ⋅dλ (2) where f(λ) is the normalized emission spectrum of the donor and ε(λ) is the absorption spectrum of the acceptor. The Förster radius (R 0 ) is given by: R 2 −4 1/ 6 0 = 0.2108 ⋅ ( J ⋅ κ ⋅ n ⋅ φ D ) (3) where the orientational factor is assumed in the dynamic isotropic regime (κ 2 = 2/3), the refractive index of the medium is n = 1.44, and φ D is the donor quantum yield. The numeric factor in Eq.(3) assumes nm units for the wavelength λ and Å units for R 0 . The Förster radius (R 0 ) of the Tryptophan(OmpF)-Ciproflaxacin donor-acceptor pair was determined to be 23.3 Å. This is an average value since each of the tryptophans has distinct fluorescence properties and therefore is expected to present different (but, in 115
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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)
Page 91 and 92: 2438 Fernandes et al. section of th
Page 93 and 94: 2440 Fernandes et al. tein oligomer
Page 95: QUANTIFICATION OF PROTEIN-LIPID SEL
Page 98 and 99: FRET Study of Protein-Lipid Selecti
Page 107 and 108: BINDING OF INHIBITORS TO A PUTATIVE
Page 109 and 110: BINDING OF INHIBITORS TO A PUTATIVE
Page 111: INTERACTION OF THE INDOLE CLASS OF
Page 114 and 115: 5272 Biochemistry, Vol. 45, No. 16,
Page 123: BINDING ASSAYS OF INHIBITORS TOWARD
Page 126 and 127: 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 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
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FIG 6. 23
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FIG. 8 A B 25
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The signalling functions of PI(4,5)
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172
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174
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zoxadiazol (NBD)-PC were obtained f
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Fig. 3. Fluorescence decays of diph
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CONCLUSIONS VII CONCLUSIONS Chapter
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CONCLUSIONS constants recovered by
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CONCLUSIONS Chapter IV ‐ Binding
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CONCLUSIONS Chapter VI - Clustering
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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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