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

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5272 Biochemistry, Vol. 45, No. 16, 2006 Fernandes et al. incorporation of the inhibitor with the concomitant increase of its effective concentration. Recently, spin-labeled derivatives of SB 242784 have been studied by electron spin resonance (ESR) (12). It was then possible to acquire information regarding the location in the membrane of the piperidine ring where the nitroxide label was inserted. However, a photophysical approach would enable a report on the whole molecule, since the indole ring is conjugated with the double bond system, and this is largely expected to dictate the inhibitor properties in the lipid phase. In this work we report a thorough study on the interaction of SB 242784 with lipid vesicles together with a detailed study of the compound behavior in aqueous solution, using for this effect its intrinsic photophysical properties (absorption, fluorescence, fluorescence anisotropy, linear dichroism), thus avoiding the derivatization of the molecule. The waterlipid partition coefficient of SB 242784 was determined, and its aggregation was studied. The position and orientation of the molecule when interacting with lipid bilayers were also obtained. The same studies were performed with another less powerful inhibitor of the same class (INH-1) to compare the results and retrieve information on the relevance of specific structural features of SB 242784 on these properties. MATERIALS AND METHODS DOPC, DOPG, and DMPC were obtained from Avanti Polar Lipids (Birmingham, AL). 5-DOX-PC and 12-DOX- PC were from Avanti Polar Lipids (Birmingham, AL). Fine chemicals were obtained from Merck (Darmstadt, Germany). All materials were used without further purification. SB 242784 and INH-1 were synthesized according to Nadler et al. (6) and Gagliardi et al. (5). Inhibitor Incorporation in Lipid Vesicles. The V-ATPase inhibitors were incorporated in lipid vesicles by two different methods: In the cosolubilization method the phospholipid and inhibitor solutions were mixed in chloroform and dried under a flow of dry N 2 . The last traces of solvent were removed under vacuum. Multilamellar vesicles were then obtained through solubilization in Tris buffer, and large unilamellar vesicles (LUVs) were produced by extrusion through polycarbonate filters with a pore size of 100 nm (13). In an alternative method of incorporation, a very small volume (
V-ATPase Indole Inhibitor Interaction with Bilayers Biochemistry, Vol. 45, No. 16, 2006 5273 The order parameters (〈P 2 〉 and 〈P 4 〉) are obtained from the equations: sin(ω)A ω Α ω)(π/2) ) 1 + 3〈P 2 〉 (1 - 〈P 2 〉)n 2 cos2 ω (3) I VH /I VV ) a sin 2 R+b (4) A ω is the absorbance at angle ω, n is the refraction index [n ) 1.5 (21)], R is the angle at which the fluorescence is measured, and the a and b parameters depend on both 〈P 2 〉 and 〈P 4 〉. Complete formalisms and further details are described in Lopes et al. (22). Dichroic ratios were determined using Glan-Thompson polarizers. In fluorescence emission measurements for determination of 〈P 4 〉, excitation and emission wavelengths were kept the same as in the remaining steady-state measurements. From the knowledge of 〈P 2 〉 and 〈P 4 〉, a combination of the maximum entropy method together with the formalism of the Lagrange multipliers is used to describe the single particle distribution function f(Ψ), where Ψ is the angle between the transition moment of the molecule and the director of the system (normal to the bilayer plane). This distribution is the broadest one that is compatible with the experimental order parameters, and to obtain the population density probability function, which is the probability of finding the transition moment between Ψ and Ψ + dΨ, it should be multiplied by sin Ψ (22). The determination of the transition moment of SB 242784 was performed for an optimized geometry of the molecule based on the density functional theory (DFT) (23), which recovered the 2Z,4E isomer that is the preponderant species (6). The theoretical level applied to the calculations was Becke3LYP/6-31G(d) (24-26). RESULTS Indole Inhibitor BehaVior in Aqueous EnVironment. Since the interaction with the membrane proceeds from the inhibitors in aqueous solution, their characterization in an aqueous environment was first carried out. Preliminary results showed both SB 242784 and INH-1 (Figure 1) to be unstable under aqueous condition (27). The fluorescence quantum yield of the two molecules in aqueous buffer was extremely low (0.01 and 0.004 for SB 242784 and INH-1, respectively) when compared with the quantum yield observed in other environments (e.g., 0.06 and 0.05 for SB 242784 and INH-1 in ethanol, respectively). The large decrease in fluorescence intensity of the inhibitors is the result of formation of nonfluorescent or “dark” aggregates, as the quantum yield values determined from transient state fluorescence measurements were larger than the ones obtained from steady-state data. This aggregation is due to the hydrophobic character of the inhibitors, and the fluorescence quenching effect is enhanced by the presence of chlorine atoms in the molecule. In the present work we measured the extent and kinetics of aggregation of both inhibitors in an aqueous environment. For this purpose, fluorescence intensity measurements were started immediately after addition of 10 µL of a ethanol stock solution to a cuvette containing 3 mL of buffer under permanent stirring, and the final concentration of inhibitor in buffer was 4 µM. The buffer used was 10 mM Tris and FIGURE 2: SB 242784 fluorescence emission self-quenching induced by aggregation in an aqueous environment. The buffer used was 10 mM Tris and 150 mM NaCl, pH ) 7.4. 150 mM NaCl, pH 7.4. The result for SB 242784 is presented in Figure 2. The kinetics for the aggregation in water of INH-1 was very fast, and the phenomenon could not be resolved as well as it was for SB 242784. In addition, the extent of aggregation, as taken from the remaining fluorescence intensity after the process reaches equilibrium, was larger for INH-1 than for SB 242784. For both inhibitors the fluorescence intensity increased dramatically upon addition of sodium dodecyl sulfate to the samples (results not shown), this suggesting disruption of aggregates and emission of monomeric inhibitors dispersed in the micellar medium and ruling out a significant contribution of photobleaching. The anisotropies of INH-1 and SB 242784 in water were 0.20 and 0.22, respectively, and did not change during the process of aggregation. This indicates that the aggregates being formed are nonfluorescent, and the residual emission of monomeric species is the one contributing to anisotropy. In an attempt to determine the existence of a critical micellar concentration (cmc) for the inhibitors in an aqueous environment, we measured fluorescence emission intensities for inhibitor solutions with concentrations up to 4 µM. A cmc should appear as a change in linearity of fluorescence intensity vs inhibitor concentration. However, a completely linear plot was obtained up to 10 nM, the limit of detection of the fluorometer (results not shown). Therefore, the aggregation offset, if existing at all, should occur for both inhibitors in water at concentrations below 10 nM, and therefore no evidence was obtained for micellar-type aggregates of these inhibitors. Inhibitor Partition to Lipid Vesicles. The fluorescence emission spectra of SB 242784 and INH-1 in the presence of DOPC vesicles are presented in Figure 3. DOPC and DOPG lipids were chosen for the reconstitution of the inhibitors because SB242784 was used by us in binding assays to selected transmembrane peptide fragments of V-ATPase that required DOPC (and in some cases a small fraction of DOPG) for correct peptide conformation while inserted in liposomes (unpublished experiments). Using the cosolubilization method for incorporation of the inhibitors (see Materials and Methods section), the fluorescence emission maximum for SB 242784 is 436 nm whereas for INH-1 it is 461 nm. On the other hand, in DOPC-DOPG (80:20 mol/mol) bilayers, the SB 242784 fluorescence
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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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Page 68 and 69: Figure I.20 - Relative binding cons
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Page 75 and 76: PROTEIN-PROTEIN AND PROTEIN-LIPID I
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Page 83 and 84: 2430 Biophysical Journal Volume 85
Page 85 and 86: 2432 Fernandes et al. Coat protein
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Page 89 and 90: 2436 Fernandes et al. FIGURE 4 (A)
Page 91 and 92: 2438 Fernandes et al. section of th
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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
Page 109 and 110: BINDING OF INHIBITORS TO A PUTATIVE
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 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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136
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dynamin. Soon after, the same group
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140
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142
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Introduction Control of membrane re
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Steady-state fluorescence measureme
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Rho-DOPE (acceptor). The increase o
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where η is the viscosity of the me
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ound conformation of the BAR domain
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19. Fernandes, F., L. M. S. Loura,
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Figure Legends Fig.1: N-terminal am
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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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