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

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central D-subunit, which in turn drives rotation of the hexameric ring of c subunits immersed in the membrane. Subunit a is held fix relative to the A3B3 hexamer by the peripherical stalk, which functions as a stator. Proton transport is hypothesized to occur at the interface between the subunit a and the subunits c, c’ and c’’: rotation of the ring of c subunits relative to subunit a, brings the buried acidic residues into sequential contact with two hemichannels in subunit a, that provide access to each side of the membrane, driving unidirectional proton transport (Fig. III-2) (Grabe et al., 2000). Figure III-1: Representation of the resorbing osteoclast and acidification of the resorption lacunae (taken from Väänanen, 2005). Figure III-2: Structural Model of V-ATPase and proposed rotary mechanism of ATP-driven proton translocation. The most important amino acid residues for proton translocation are marked (adapted from Nishi and Forgac, 2002) 80
BINDING OF INHIBITORS TO A PUTATIVE BINDING DOMAIN OF V-ATPase In eukaryotic cells, V-ATPase, apart from being present in the plasma membrane of osteoclasts and kidney acid secreting cells, is also found in many organelles such as vacuoles, lysossomes, coated vesicles, Golgi, and secretory vesicles. Numerous physiological processes depend on the activity of V-ATPases, including intracellular targeting, protein processing, transport of metabolites, receptor-mediated endocytosis, neurotransmitter uptake, and apoptosis (Finbow and Harrison, 1997; Nishi and Forgac, 2002). The macrolide antibiotics bafilomycins and concanamycins are specific, highly potent inhibitors of all eukaryotic V-ATPases in vitro and in vivo (Fig. III-3). However, their unselective action towards V-ATPases turns them to be extremely toxic agents and unsuitable for therapeutical use in osteoporosis treatment (Dröse and Altendorf, 1997). Recently, novel and selective inhibitors of osteoclasts V-ATPase have been synthesized according to structure-function relationships of bafilomycins derivatives (Gagliardi et al., 1998a; Gagliardi et al. 1998b), suggesting that selective modulation of different V- ATPases might be possible by this new indole class of inhibitors, namely by its most promising representative SB 242784 (INH-3) (see Fig. III.3) (Nadler et al., 1998). SB 242784 studies with animal models of bone resorption showed a remarkable efficiency in the prevention of ovariectomy-induced bone loss on rats, and no adverse effects of its administration on the animals was observed. In situ studies revealed that SB 242784 inhibited osteoclastic V-ATPase activity at 1000 times smaller concentrations than enzymes from any other tissue evaluated (Visentin et al., 2000). Figure III-3: V-ATPase inhibitors. 81
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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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Page 72 and 73: In Figure I.21, theoretical simulat
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 95: QUANTIFICATION OF PROTEIN-LIPID SEL
Page 98 and 99: FRET Study of Protein-Lipid Selecti
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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
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Page 140 and 141: Introduction Quinolones are broad-s
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Page 150 and 151: APPENDIX - Derivation of the FRET r
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Page 154 and 155: [14] L. Plançon, M. Chami, L. Lete
Page 156 and 157: acceptors) (Eqs. 4-6) (⋅-⋅-⋅)
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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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