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

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5276 Biochemistry, Vol. 45, No. 16, 2006 Fernandes et al. FIGURE 7: Orientation distribution function in DMPC of SB 242784 (- - -) and INH-1 (s). FIGURE 8: Proposed topography and orientation of SB 242784 in a DOPC bilayer. The molecule is depicted from the estimated most probable angle and depth (see Discussion). to result in a smaller hydrophilicity, but this does not seem to be the case. Regarding the extent of aggregation, one possibility is that the piperidine ring induces a different type of packing in SB 242784 aggregates which quenches the molecule fluorescence less efficiently than in INH-1 aggregates, resulting in partially fluorescent aggregates. For INH-1 aggregates it is reasonable to assume that these would consist of stacked molecules with π-π interactions, but for SB 242784, this type of π-π stacking would not be able to exclude the piperidine ring from the aqueous environment and another aggregation geometry would have to come into play. The larger self-quenching of INH-1 in water would then not be solely dictated by the extent of aggregation but also related to a more effective geometry of packing regarding the quenching of fluorescence. The absence of a micellar type of aggregate for these inhibitors is most probably related to the absence of significative amphiphilic character. Both inhibitors partition with a high efficiency to lipid vesicles, and for SB 242784 the partition coefficient is independent of the method of incorporation (Table 1). Nevertheless, the partition to the lipid phase is only complete at moderately high lipid concentrations (for a K p ≈ 12000, 95% of the total inhibitor population is incorporated only at a lipid concentration of 2 mM). For INH-1, the molecule partitions slightly more effectively when added to the preformed liposomes, but the differences in the partition coefficients are close to the uncertainty of the measurements. On thermodynamic grounds the two partition coefficients should in fact be identical upon reaching equilibrium. However, in most situations, the one from cosolubilization is higher than adding the solute from the aqueous solution, this being the case among other situations when stable aggregates (e.g., micelles) are formed in water. The identical values observed for the two methodologies of incorporation are consistent with the absence of a cmc previously described. The lower value for the partition coefficient of SB 242784 in DOPC-DOPG bilayers indicates a decreased partition to the bilayer upon the change in headgroup composition. This effect will be addressed in detail below when the fluorescence emission shifts of the inhibitors are discussed. INH-1 anisotropy was smaller when it was added to preformed vesicles (〈r〉 ) 0.24) when compared with the one observed for the same molecule incorporated via cosolubilization (〈r〉 )0.31), and this is likely to be related to energy migration inside aggregates in the membrane. As there is an overlap between the absorption and fluorescence emission spectra of the inhibitors (Figure 3), energy migration (energy homotransfer) is operative for two molecules of inhibitor lying in close proximity as in the case of an aggregate. The INH-1 Förster radius (R 0 )(16) for energy migration while in lipid membranes is 11 Å (result not shown), and therefore for INH-1 in close proximity fluorescence depolarization is expected. The effects of energy migration can only be observed via the fluorescence anisotropy which is expected to decrease, since the fluorescence lifetime remains constant in this type of photophysical interaction. At variance with the aggregates in water, these aggregates must be fluorescent; i.e., there is no selfquenching, as in order to change the steady-state anisotropy they have to contribute to a significant fraction of the total fluorescence. This feature is probably related to the specific geometry of the aggregate in the membrane. In addition, in the case that quantitative dimer formation is invoked, the intermolecular distance would be 12.7 Å as concluded from the theoretical formalism (32); this is slightly larger than the distance for a collisional complex, so this would mean that no contact (static) fluorescence quenching would happen. Possibly, INH-1, when added to preformed vesicles, either (i) segregates to a membrane-perturbed region, and what is being observed is not strictly aggregate formation but increased probability of insertion in a membrane-perturbed area, leading to a higher local monomer concentration, such as observed for micelles (33) and membranes (34), or (ii) is only partially aggregated (with intermolecular distance
V-ATPase Indole Inhibitor Interaction with Bilayers Biochemistry, Vol. 45, No. 16, 2006 5277 structural differences of the two molecules (presence of the piperididine ring in SB 242784). Inhibitor TransVerse Location and Orientation in Lipid Vesicles. The large blue shifts observed upon the incorporation of the inhibitors in lipid vesicles are an indication that the inhibitors are not adsorbed to the surface of the bilayer but are somehow buried, having some contact with the acyl chain region. The wavelengths of maximum fluorescence emission for both inhibitors (436 and 461 nm) are almost the same as the ones observed for the same molecules in acetone (27). Taking as a reference the dielectric constant of acetone (ɛ ) 20.7), the corresponding region of PC bilayers with the same polarity properties lies in the headgroup region, outside the hydrocarbon core of the bilayer (35, 36), which for DOPC can be roughly located at >14 Å from the center of the bilayer (29). On the other hand, SB 242784 in solution only exhibited such a blue-shifted emission when solubilized in nonprotic solvents, and as such this inhibitor is likely not to be in contact with a high density of water molecules when in lipid bilayers, excluding the possibility of a strictly superficial position, and therefore a positioning in the polar/apolar interface of the bilayer is more likely. Although other factors such as hydrogen bonding might affect the emission properties of the fluorophores, it is worthwhile noting that in addition to both inhibitors having fluorescence properties almost identical to those observed in acetone, they are located almost at the same distance to the center of the bilayer, as observed by acrylamide quenching and the parallax method, the latter positioning SB 242784 and INH-1 at 12.8 and 11.9 Å from the center of the bilayer (corresponding approximately to the position of the fourth to third carbon of the DOPC acyl chain in the fluid state), respectively. The difference in the position between the two inhibitors is
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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
Page 68 and 69: Figure I.20 - Relative binding cons
Page 70 and 71: 2.9. Lipid phase preferential parti
Page 72 and 73: In Figure I.21, theoretical simulat
Page 75 and 76: PROTEIN-PROTEIN AND PROTEIN-LIPID I
Page 77 and 78: PROTEIN-PROTEIN AND PROTEIN-LIPID I
Page 79: PROTEIN-PROTEIN AND PROTEIN-LIPID I
Page 83 and 84: 2430 Biophysical Journal Volume 85
Page 85 and 86: 2432 Fernandes et al. Coat protein
Page 87 and 88: 2434 Fernandes et al. leads to an a
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
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
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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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Page 166 and 167: 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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