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

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placement of the protein around the tryptophan is no longer symmetrical as for Trp 61 , and energy transfer must be accounted differently: 2 2 ⎡ ⎧ ⎡ wi / wi + Rp 6 ⎪ 2 ⎪ ⎧ ⎡⎛ t ⎞⎛ R ⎞ ⎤ 0 6 ⎪ ⎫ ρ nonbound '() t = ⎢ ∏ exp −2π n2w ⎢ i 1−exp ⎢ − α ⎥ f( α) dα − ⎢ ⎨ ⎨ ⎬ i ⎢ ∫ ⎜ ⎟⎜ ⎟ τ 2 '2 ⎢ wi / w D w i ⎥ ⎢ ⎪ i + R ⎪ ⎝ ⎠⎝ ⎠ ⎪ d ⎣ ⎩ ⎣ ⎩ ⎣ ⎦ ⎭ ⎡ ⎤ ⎤⎫⎤ ⎛ ⎞ ⎪⎥ ⎜ ⎟ ⎥ ⎣ ⎦ ⎦⎭⎦⎥ 1 6 t ⎛ R ⎞ 0 6 −3 exp α f( α) α dα⎥ ∫ ⎢ − ⎜ ⎟ ⎥ ⎬ τ 2 2 ⎢ wi / w D wi ⎥ ⎥ i + R ⎝ ⎠⎝ ⎠ ⎪ p (6) ρ nonbound’ is therefore the FRET contribution to the decay of a donor located in the surface of a protein of radius R P due to the presence of acceptors randomly distributed in i different planes (for details on the derivation of this component and on function f(α) see Appendix). Using eqs. (1) and (4)–(6), theoretical expectations for FRET efficiencies for a random distribution of acceptors can be estimated. In the simulations and data analysis of FRET efficiencies it was considered that Trp 61 and Trp 214 contributed equally to the fluorescence intensity of the protein. Also, energy migration among the Trp 61 does not affect the geometry of the problem, and energy migration between Trp 61 and Trp 214 is a very inefficient process due to the distance (30 Å) between the two residues. Contribution to FRET of specifically bound acceptors. To quantify the contribution of specifically bound CP to FRET, it is first necessary to relate the binding constant of this antibiotic and OmpF (K B ) to the factor γ (fraction of OmpF bound to CP) in Eq. 4: ( 1 [ OmpF] KB [ CP] KB) ⎡ + ⋅ + ⋅ + ⎤ γ = ⎢ ⎥ 2⋅K 2 B ⋅ OmpF ⎢ 2 ( 1+ [ OmpF] ⋅ KB + [ CP] ⋅KB) −4⋅KB ⋅[ OmpF] ⋅[ CP ⎥ ⎣ ] ⎦ [ ] Total (7) where [OmpF] Total and [OmpF] are the total and unbound concentrations of OmpF, and [CP] is the concentration of unbound CP. It is assumed that only 1:1 complexes can be 118
BINDING OF A QUINOLONE ANTIBIOTIC TO BACTERIAL PORIN OmpF formed between OmpF and CP. The 1:1 complex model is supported by data from a previous study [25]. In the event of binding of CP to Ompf, the fluorescence of the several Trp’s present in one trimer will be quenched differently, as the distances between them and the antibiotic will be also different. Therefore, also for ρ bound , two different contributions for Trp 214 and Trp 61 must be considered. ρ = ρ + ρ bound bound (Trp 214) bound (Trp61) (8) Assuming that at least one of the tryptophans of OmpF is part of a hypothetical CP binding site and subsequently completely quenched, and that no more than one antibiotic molecule can be specifically bound to a OmpF trimer at a given time, two models were devised: i) the CP binding site is close to the center of the trimer and binding of antibiotic leads to complete quenching of the three Trp 61 (distance of Trp 61 to CP
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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
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
Page 142 and 143: [9-12]. Having this into considerat
Page 144 and 145: any case, very similar, probably wi
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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Page 188 and 189: FIG. 3A FIG. 3B 19
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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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