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

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any case, very similar, probably within 1-2 Å of each other) Förster radii for energy transfer to CP. Through an analysis of the extent of fluorescence quenching of a donor by the acceptor in a FRET experiment it is possible to calculate a binding constant (K B ) between these two molecules. However, care must be taken when working in solutions where acceptors are highly concentrated, as it is often the case in experiments performed on liposomes. In these cases, when both donors and acceptors partition to the lipid bilayer and interact there, the concentration of acceptors around the donors increases dramatically relatively to a situation where they are both free in solution. FRET can be efficient at distances up to 100 Å depending on the Förster radius of the donor-acceptor pair [19] and therefore, donors in a medium highly concentrated in acceptors, besides being susceptible to fluorescence quenching due to formation of specific donor-acceptor complexes, are also quenched by non-bound, nearby acceptors. Only the first contribution is relevant for the determination of the binding affinity of the donoracceptor complex, and as the two contributions are not additive (Eq. 4), formalisms that accurately account for the contribution of non-bound acceptors for the experimentally measured energy transfer efficiencies must be derived, considering the geometrical peculiarities of the studied system. The donor fluorescence decay in the presence of acceptors (i DA (t)) is described as: i () t = γ ⋅i () t ⋅ρ () t ⋅ρ () t DA D bound nonbound ( γ ) + 1- ⋅i ( t) ⋅ρ () t D nonbound (4) where γ is the fraction of OmpF bound to CP, ρ bound is the FRET contribution from energy transfer to acceptors bound to Ompf, and ρ nonbound is the FRET contribution arising from energy transfer to non-bound acceptors, randomly distributed in different planes (i) from the donors. Contribution to FRET of non-bound acceptors. ρ nonbound for a cylindrically symmetric donor geometry is given by [20]: 116
BINDING OF A QUINOLONE ANTIBIOTIC TO BACTERIAL PORIN OmpF ⎡ wi ⎛ ⎞⎤ 2 2 ⎢ wi + R ⎜ e 6 2 1−exp( −t⋅b (5) i ⋅α ) ⎟⎥ ρnonbound = ∏ ⎢exp⎜−2⋅n2 ⋅π⋅wi ⋅ dα⎟⎥ 3 i ⎢ ∫ α 0 ⎥ ⎢ ⎜ ⎟ ⎣ ⎝ ⎠⎦ ⎥ where b i =(R 2 0 /w i ) 2 τ −1/3 D , n 2 is the acceptor density in each bilayer leaflet (number of acceptors per unit area), w i is the distance between the plane of the donors and the i-th plane of acceptors and R e is the donor exclusion radius (which defines the area around the donor from where the acceptors are excluded). In protein-ligand FRET studies the exclusion radius is particularly important if the size of the protein is comparable to the Förster radius of the donor-acceptor pair. In order to calculate ρ nonbound , a model must be used to describe the positions of the donors and acceptors in the bilayer. OmpF has two clear belts of aromatic residues at the lipid/water interface separated by 25 Å [21]. Both Trp’s (61 and 214) are located in the same aromatic belt and are assumed to be in the same plane, at 12.5 Å from the center of the bilayer. When incorporated in liposomes, CP is located in the headgroup region of the bilayer [22] and according to Nagle and Tristan-Nagle [23] for DMPC the headgroups average position is 18 Å away from the centre of the bilayer. In the FRET simulations this is considered to be the position of the plane of acceptors and w 1 and w 2 are assumed to be 5.5 Å and 30.5 Å (Fig. 4A). In our formalism we consider that there is no partition of non-bound CP in the bilayer area occupied by the OmpF trimer apart from the specifically bound population of antibiotic molecules. Trp 61 is located at the trimer interface (Fig. 4B) and the exclusion area for non-bound CP will be significant. R e (Eq. 5) of Trp 61 is assumed to be 30 Å which is the approximate diameter of an OmpF monomer in the bilayer plane [24] (Fig. 4B). On the other hand, Trp 214 is located in the periphery of the OmpF trimer and the distribution of non-bound CP around it will be cylindrically asymmetric. This difference in the distribution of acceptors sensed by the two donors (Trp 214 and Trp 61 ) can only be accounted through the use in the FRET simulations of two different ρ nonbound contributions. For Trp 61 the FRET contribution is simply given by Eq.5 and setting i = 2, w 1 = 5.5 Å, w 2 = 30.5 Å and R e = 30 Å. In the case of Trp 214 a large section of the bilayer is also occupied by the protein trimer itself and inaccessible to acceptors. However, this 117
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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
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
Page 136 and 137: apparently the most significant as
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Page 140 and 141: Introduction Quinolones are broad-s
Page 142 and 143: [9-12]. Having this into considerat
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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Page 188 and 189: FIG. 3A FIG. 3B 19
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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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