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

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⎛ II t ⎛ R ⎞ 0 ρ () t = exp − bound(Trp 214) ⎜ ⋅⎜ ⎟ τ D d ⎝ ⎝ 2 ⎠ 6 ⎞ ⎟ ⎠ (12) Results and Discussion Successive additions of small volumes of a CP solution to OmpF proteoliposomes resulted as expected in a decrease of fluorescence from OmpF (Fig.6). Using Eqs 4-6 it is possible to compare the theoretical expectation for the efficiencies of energy transfer from the tryptophans of OmpF to unbound CP (randomly distributed) with the data obtained experimentally. Clearly the extent of quenching of the tryptophans cannot be exclusively explained on the basis of energy transfer to unbound CP, and a FRET contribution to specifically associated antibiotic must be considered. The binding constants that allow for better fits to the data points are different depending on the model for binding that is considered (I or II). In the case of model I, that assumes binding of CP close to the centre of the trimer, quenching is much more effective as the binding site is in the vicinity of three tryptophans (Trp 61 ), while for model II only one tryptophan is located near the bound antibiotic. This results in the recovery of larger K B values for model II than for model I. Upper and lower limits for K B (that allow respectively for better fits to the data points corresponding to the lower and higher CP concentration ranges) were determined for each model, and are shown in Table I. In a previous study, changes in CP absorption spectra were used to estimate a OmpF-CP binding constant and values of log(K B ) = 3.85 ± 0.34 or 4.17 ± 0.03 were obtained depending on the method chosen to analyze the absorption spectra changes after addition of OmpF in a micellar environment [25]. These values fall within or close to the range of K B obtained by us from the energy transfer data assuming binding of CP to the periphery of OmpF (3.58 < K B < 4.00). This agreement with an alternative method can be seen as an indication that the antibiotic binding site is likely to be located away from the centre of the OmpF trimer where FRET would be more efficient and imply a smaller K B . 120
BINDING OF A QUINOLONE ANTIBIOTIC TO BACTERIAL PORIN OmpF Conclusions In the present study, the CP fluoroquinolone is shown to associate with OmpF using FRET methodologies. The high symmetry of the OmpF trimer allowed the analysis of FRET data with formalisms that accounted for the different distributions of acceptors around the two types of tryptophans present in the protein. This methodology is suitable for application in the analysis of FRET data obtained with similar protein systems and can be adapted to different geometries. The new FRET formalisms presented here for a donor in the surface of a cylinder of large radius (with radius comparable to the Förster radius) are expected to be of great assistance in the analysis of FRET data obtained with membrane proteins presenting donor fluorophores in the protein-lipid interface. The equilibrium constant retrieved for the association event between OmpF and CP assuming binding of the antibiotic in the periphery of the porin is in agreement with a value determined previously by an independent method (in a micellar environment), indicating that the binding site of the antibiotic is likely to be located away from the center of OmpF. Also, the FRET methodology can be applied in cases where there are no spectral shifts, and its sensitivity is higher as compared to absorption methodologies. 121
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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
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
Page 138 and 139: 110
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 146 and 147: placement of the protein around the
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
Page 164 and 165: 136
Page 166 and 167: dynamin. Soon after, the same group
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Page 170 and 171: 142
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
Page 186 and 187: FIG. 1A FIG.1B 17
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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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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