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

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Our understanding of the mechanism of V-ATPase inhibition is rudimentary. Nevertheless, site-directed mutagenic studies strongly supported the hypothesis of the location of the bafilomycin A 1 binding site in the c-subunit of V-ATPase (Bowman and Bowman, 2002; Bowman et al., 2004). These studies indicated several amino acid residues in the c-subunit of V-ATPase for which mutations induced resistance to bafilomycin A 1 . The majority of these residues were located at the 4 th putative TM helix, and four of them (L132, F136, L140, and Y143) are positioned in the same helix side, which is expected to be exposed to the lipid environment (Harrison et al., 2000). Some residues found in other domains of the c-subunit are apparently also relevant for bafilomycin binding. However, it is not clear, if they are part of the bafilomycin binding site, as a structure formed by these residues would imply a helix organization for the c- subunit which is in disagreement with the one suggested by cysteine cross-linking experiments (Harrison et al., 1999). Another study detected immobilization of spinlabeled residues of c-subunit in the presence of concanamycin A, in agreement with a binding site location on this region of V-ATPase (Páli et al., 2004). Whyteside et al. (2005) found that SB242784 bound competitively with concanamycin to the c-subunit of V-ATPase. However, a mutation on Y143 from the c- subunit of V-ATPase on yeast conferred different degrees of resistance for concanamycin and SB242784, suggesting that the binding sites for these inhibitors are overlapping but not identical (Páli et al., 2004). Subunit c and c´ present very similar sequences. However, mutations in subunit c´ homologous to the ones in subunit c that conferred resistance to bafilomycin have no effect (Bowman et al., 2004). This fact, led to the proposal that either subunit c´ does not present bafilomycin binding sites or that the affinity of the macrolide antibiotic binding site was much smaller in subunit c´. One strategy to resolve the problem of the binding site location for the above mentioned inhibitors is the use of a reductionist approach. Reductionist approaches to protein-ligand problems through use of peptides have been used in the past with success and peptides comprising entire binding sites were already shown to present ligand binding affinities identical to the ones observed with the intact protein (Lentz et al., 1998; Wilson et al., 1985; Neumann et al., 1986; Taylor et al., 1995; Ried et al., 1996; Scatigno et al., 2004). 82
INTERACTION OF THE INDOLE CLASS OF V-ATPase INHIBITORS WITH LIPID BILAYERS 2. INTERACTION OF THE INDOLE CLASS OF VACUOLAR H + -ATPase INHIBITORS WITH LIPID BILAYERS 83
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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
Page 60 and 61: thickness of the bilayer (Section 1
Page 62 and 63: some α-helical membrane proteins a
Page 64 and 65: The formation of a lipid population
Page 66 and 67: 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
Page 93 and 94: 2440 Fernandes et al. tein oligomer
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: BINDING OF INHIBITORS TO A PUTATIVE
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 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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FIGURE 4A FIGURE 4B 132
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FIGURE 6A FIGURE 6B 134
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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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