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

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apparently the most significant as the efficiency of the quinolones is not directly related to the water/lipid partition coefficient of the molecules (Vazquez et al., 2001). Indeed, by either not expressing or expressing structurally changed outer membrane porins, entrance of quinolones in bacteria is severely impaired (Mascaretti, 2003; Chevalier et al., 2000). OmpF is one of the porins involved in the permeation process of quinolones across the outer membrane of bacteria (Chapman and Georgopapadakou, 1988). It was the first membrane protein to yield crystals of size and order allowing to be analyzed by X-ray crystallography (Garavito and Rosenbusch, 1980). This porin forms a homotrimer in the outer membrane and each monomer presents β-barrel structure in which one of the loops folds back to the barrel, forming a constriction zone at half the height of the channel (Figure 1 in section 2 of this Chapter). This loop is expected to contribute significantly to the control of the permeability of OmpF. It is not yet know whether OmpF operates as a channel or as an enabler of quinolones diffusion across the lipid-OmpF interface. Therefore, the knowledge of the exact type of interaction established by OmpF and quinolones is of great biological relevance, and could be potentially useful in the development of more effective drugs in the future. In this chapter, the interaction of OmpF and a second generation quinolone, ciprofloxacin (CP), was studied. CP is a 6-fluoroquinolone which structure is shown in Figure IV-1. Recent studies suggested a 1:1 stoichiometry for OmpF-CP interaction (Neves et al., 2005). Figure IV-1: Chemical structure of CP. 108
BINDING OF A QUINOLONE ANTIBIOTIC TO BACTERIAL PORIN OmpF 2. CIPROFLOXACIN INTERACTIONS WITH BACTERIAL PROTEIN OMPF: MODELLING OF FRET FROM A MULTI-TRYPTOPHAN PROTEIN TRIMER ABSTRACT The outer membrane protein F (OmpF) is known to play an important role in the uptake of fluoroquinolone antibiotics by bacteria. In this study, the degree of binding of the fluoroquinolone antibiotic ciprofloxacin to OmpF in a lipid membrane environment is quantified using a methodology based on Förster resonance energy transfer (FRET). Analysis of the fluorescence quenching of OmpF is complex as each OmpF monomer presents two tryptophans at different positions, thus sensing two different distributions of acceptors in the bilayer plane. Specific FRET formalisms were derived accounting for the different energy transfer contributions to quenching of each type of tryptophan of OmpF, allowing the recovery of upper and lower boundaries for the ciprofloxacin-OmpF binding constant (K B ). log (K B ) was found to lie in the range 3.15-3.62 or 3.58-4.00 depending on the location for the ciprofloxacin binding site assumed in the FRET modelling, closer to the centre or to the periphery of the OmpF trimer, respectively. This methodology is suitable for the analysis of FRET data obtained with similar protein systems and can be readily adapted to different geometries. 109
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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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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
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Page 111: INTERACTION OF THE INDOLE CLASS OF
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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 150 and 151: APPENDIX - Derivation of the FRET r
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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
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Page 176 and 177: Rho-DOPE (acceptor). The increase o
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FIG. 1A FIG.1B 17
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FIG. 3A FIG. 3B 19
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FIG.5A 21
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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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