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

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CONCLUSIONS constants recovered by FRET were almost identical to the ones recovered for an aggregated form of mcp, confirming the reliability and sensitivity of the technique. mcp showed affinity for lipids with acyl chain lengths that matched the hydrophobic length of its TM domain, and for anionic lipids, particularly PA and PS. Only the lipids in the immediate vicinity of the protein are sufficiently immobilized to be detected by ESR, and as a consequence the binding affinity determined by ESR only refers to this shell of lipids. On the other hand, FRET is not limited to this, and for high Förster radii such as the one for the donor-acceptor pair used in this study, the efficiency of FRET from the protein to an acceptor in the second shell of lipids is similar to the efficiency of FRET to an acceptor in the first shell around the protein. In this way, the compatibility of the results from FRET and ESR points clearly to the validity of the annular model for protein-lipid interaction (single TM protein only induces changes in lipid distribution on the first shell of lipids around it). This FRET methodology for the quantification of protein-lipid selectivity also offers the possibility of probing different shells of lipids around the protein by choosing donor-acceptor pairs with different Förster radii. To increase the sensitivity of the method to selectivity in the first shell of lipids around the protein, a small Förster radius would be advisable, that allowed to concentrate the majority of the contributions to donor quenching on the acceptors located there. 183
Chapter III ‐ Binding of Inhibitors to a Putative Binding Domain of V‐ATPase. The studies conducted allowed us to know in detail the properties of the indole type of inhibitors in lipid membranes. The molecules studied, which correspond to the initial (low efficiency) and final stage (high efficiency) in the development of these inhibitors as potential drugs in osteoporosis treatment, present almost identical properties in the liposomes environment. The preference of the lipid phase over the aqueous phase, the position in the bilayer and the orientation of the molecules were all almost identical for both molecules studied. According to the results of Gagliardi et al. (1998a), the change in inhibition efficiency of the V-ATPase inhibitors within the indole class presented some extent of correlation to the hydrophobicity of the molecules, i.e. the more hydrophobic indole inhibitor molecules had also a tendency to be more efficient inhibitors of V-ATPase. It was possible that the interaction of the molecules with the lipid milieu was significantly different within this class of inhibitors, and this could correspond to dramatically different efficiencies of inhibition, explaining the 10 3 difference in IC 50 values of INH-1 and SB242784. In this way, the process of inhibitor binding to the lipid bilayer would be crucial to the efficiency of the inhibition event. However, the results obtained point to a non-determinant role of inhibitor interaction with the lipid bilayer, and the specific molecular recognition processes with the enzyme’s inhibition site are very likely to be responsible for dictating the large differences of inhibition efficiencies within the V- ATPase indole inhibitors class. The binding studies of bafilomycin and SB242784 to the peptide corresponding to the 4 th TM helix of subunit c of V-ATPase revealed no affinity at all for SB242784 and very low affinities for bafilomycin. These results can be seen as an indicator that the binding pocket for the inhibitors is not solely composed of the lipid exposed face of the 4 th TM helix of subunit c, but also involves contributions of amino acids from other domains of the protein. This proposal is in agreement with the hypothesis of the inhibitors operating as a stone in a gear, blocking movements of the TM domains of subunit c and interrupting proton transport. 184
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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
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FRET Study of Protein-Lipid Selecti
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BINDING OF INHIBITORS TO A PUTATIVE
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BINDING OF INHIBITORS TO A PUTATIVE
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INTERACTION OF THE INDOLE CLASS OF
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5272 Biochemistry, Vol. 45, No. 16,
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BINDING ASSAYS OF INHIBITORS TOWARD
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1778 F. Fernandes et al. / Biochimi
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apparently the most significant as
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110
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Introduction Quinolones are broad-s
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[9-12]. Having this into considerat
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any case, very similar, probably wi
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placement of the protein around the
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⎛ II t ⎛ R ⎞ 0 ρ () t = exp
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APPENDIX - Derivation of the FRET r
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2 2w J ( t) = '2 R − R + w / 1
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[14] L. Plançon, M. Chami, L. Lete
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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
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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
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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Page 202 and 203: zoxadiazol (NBD)-PC were obtained f
Page 204 and 205: Fig. 3. Fluorescence decays of diph
Page 206 and 207: CONCLUSIONS VII CONCLUSIONS Chapter
Page 210 and 211: CONCLUSIONS Chapter IV ‐ Binding
Page 212 and 213: CONCLUSIONS Chapter VI - Clustering
Page 214 and 215: FINAL CONSIDERATIONS AND PROSPECTS
Page 216 and 217: BIBLIOGRAPHY IX BIBLIOGRAPHY Albert
Page 218 and 219: BIBLIOGRAPHY Phosphatidylinositol 4
Page 220 and 221: BIBLIOGRAPHY Glaubitz, C., Grobner,
Page 222 and 223: BIBLIOGRAPHY Koehorst, R. B. M., Sp
Page 224 and 225: BIBLIOGRAPHY Mishra, V. K., Palguna
Page 226 and 227: BIBLIOGRAPHY Pluschke, G., Hirota,
Page 228 and 229: BIBLIOGRAPHY Sperotto, M. M., and M
Page 230: BIBLIOGRAPHY Williamson, I. M., Alv
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