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

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1784 F. Fernandes et al. / Biochimica et Biophysica Acta 1758 (2006) 1777–1786 Fig. 7. Fluorescence emission quenching of Tyr15 of peptide H4 by FRET to SB 242784 (●). Curve corresponds to the theoretical expectation for FRET in the absence of inhibitor binding to peptide H4 (see text). The range of SB 242784 to total phospholipid ratios in these experiments was 0.18% to 1.8%. Inset: overlap between tyrosine fluorescence emission ( ) and SB242784 absorption spectrum (–––), R 0 (Tyr-SB 242784)=24 Å. When the FRET binding assay was applied to the peptide H4/SB 242784 system, the results were substantially different (Fig. 7). With this inhibitor, the obtained FRET efficiencies matched the theoretical expectations for a random distribution of acceptors, suggesting absence of binding between SB 242784 and the peptide. No uncertainty exists regarding this result as the position of the inhibitor inside the bilayer is accurately know from parallax fluorescence quenching techniques (12.8 Å from the center of the bilayer — [36]). 5. Discussion 5.1. Use of peptide H4 as a model peptide for the 4th transmembrane segment of subunit c Aggregation of transmembrane peptides containing a hydrophobic sequence and flanked by basic residues has already been reported [38–40], thus detection of aggregates of peptide H4 at low L/P ratios (
F. Fernandes et al. / Biochimica et Biophysica Acta 1758 (2006) 1777–1786 1785 [26]. In the same way, c-subunit residues shown to be important for bafilomycin A 1 inhibitory activity through mutational studies [9] could not be directly involved in the binding of the inhibitor, but still play a role in the inhibition mechanism. Here, we proposed to further define the structural requirements for bafilomycin A 1 and SB 242784 binding to the V-ATPase, by focusing on the protein domain expected to be the primary contributor to the inhibitor binding site, the putative 4th transmembrane domain of the c-subunit. The extent of binding efficiencies achieved using only peptides corresponding to this section of the protein is an indicator of the relevance of the remaining protein domains to the binding of inhibitor, the first step in the inhibitory mechanism. No evidence was found supporting SB 242784 association with the peptide H4. On the other hand, bafilomycin A 1 was shown to possess only moderate affinity for the transmembrane peptides. Due to uncertainties in bafilomycin A 1 position in bilayers it was impossible to rigorously quantify a binding constant for the process, but a reliable range for this parameter could be presented. The differences in bafilomycin A 1 binding efficiencies for the peptide H4, H4c′ and H4 A ← E are not significant as they fall within, or close to the error of the fits. For H4c′ this result is somewhat surprising. Mutations on conserved residues of the c-subunit known to confer resistance to bafilomycin A 1 and concanamycin were shown to be ineffective in the c′ and c′ isoforms [9]. It was proposed that if a bafilomycin A 1 binding site was present on these isoforms, then it must possess lower affinity than in the c isoform [9]. If this is the case, then the differences in binding efficiencies between the two isoforms must be explained by discrepancies in the interaction of the inhibitor with parts of the protein other than the highly conserved putative 4th transmembrane helix. Although the contribution from the 4th transmembrane segment (TM4) to the binding site affinity for bafilomycin A 1 is not negligible, it is absolutely unable to explain the extremely low IC 50 of bafilomycin A 1 (0.1 nM). The binding efficiencies observed for concanamycin (IC 50 close to bafilomycin A 1 ) and the intact isolated c-subunit [27] were more than 1000 times larger than the ones recovered in the present study. Therefore, it is clear that either: (i) the other transmembrane segments from the c-subunit, namely TM1 and TM2, are essential in the formation of an efficient inhibitor binding site, or that (ii) interactions of TM4 with other protein segments are required for this protein section to assume the appropriate folding which enables the establishment of the necessary interactions for an efficient binding between the residues of the 4th transmembrane segment of the c-subunit and the inhibitors. Absence of SB 242784 binding to the peptide H4 could be explained by the much lower IC 50 of this inhibitor (26 nM in chicken osteoclasts [22]) that reflects a much lower binding site affinity for this inhibitor, and the absence of remaining c-subunit transmembrane segments potentiates this effect. In this work, in addition to the detailed comparison of the different inhibitors and peptides, it was concluded that in contrast to some reported cases [11–16], there is a very significant difference in binding when comparing the binding to selected protein segments or to the whole protein. Although studies using peptides are very relevant, clearly in the case of the interaction between the bafilomycin A 1 or SB 242784 with the c-subunit from V-ATPase, the whole protein architecture and the environment that it provides, are key factors for an efficient binding. Acknowledgments This work was supported by contract no. QLG-CT-2000- 01801 of the European Commission (MIVase consortium). M. H. and M. P. are members of the COST D22 Action of the European Union. F. F. acknowledges grant SFRH/BD/ 14282/2003 from FCT (Portugal). This work was partially funded by FCT (Portugal) under the program POCTI. References [1] M.E. Finbow, M.A. Harrison, The vacuolar H+-ATPase: a universal proton-pump of eukaryotes, Biochem. J. 324 (1997) 697–712. [2] T. Nishi, M. Forgac, The vacuolar (H+)-ATPases—Nature's most versatile proton pumps, Mol. Cell. Biol. 3 (2002) 94–103. [3] B. Powell, L.A. Graham, T.H. Stevens, Molecular characterization of the yeast vacuolar H+-ATPase proton pore, J. Biol. Chem. 275 (2000) 23654–23660. [4] M.A. Harrison, M.E. Finbow, J.B. Findlay, Postulate for the molecular mechanism of the vacuolar H(+)-ATPase (hypothesis), Mol. Membr. Biol. 14 (1997) 1–3. [5] B.J. Bowman, E.J. Bowman, Mutations in subunit c of the vacuolar ATPase confer resistance to bafilomycin and identify a conserved antibiotic binding site, J. Biol. Chem. 277 (2002) 3965–3972. [6] G. Werner, H. Hagenmaier, H. Drautz, A. Baumgartner, H. Zähner, Metabolic products of microorganisms. Bafilomycins, isolation, chemical structure and biological activity, J. Antibiot. 37 (1984) 110–117. [7] M. Harrison, B. Powell, M.E. Finbow, J.B.C. Findlay, Identification of lipid-accessible sites on the Nephrops 16-kDa proteolipid incorporated into a hybrid vacuolar H+-ATPase: site-directed labeling with n-(1-pyrenyl) cyclohexylcarbodiimide and fluorescence quenching analysis, Biochemistry 39 (2000) 7531–7537. [8] T. Páli, G. Whyteside, N. Dixon, T. Kee, S. Ball, M.A. Harrison, J.B.C. Findlay, M.E. Finbow, D. Marsh, Interaction of inhibitors of the vacuolar H+-ATPase with the transmembrane Vo-sector, Biochemistry 43 (2004) 12297–12305. [9] E.J. Bowman, L.A. Graham, T.H. Stevens, B.J. Bowman, The bafilomycin/concanamycin binding site in subunit c of the V-ATPases from Neurospora crassa and Saccharomyces cerevisiae, J. Biol. Chem. 279 (2004) 33131–33138. [10] M. Harrison, J. Murray, B. Powell, Y. Kim, M.E. Finbow, J.B.C. Findlay, Helical interactions and membrane disposition of the 16-kDa proteolipid subunit of the vacuolar H1-ATPase analyzed by cysteine replacement mutagenesis, J. Biol. Chem. 274 (1999) 25461–25470. [11] T.L. Lentz, V. Chaturverdi, B.M. Conti-Fine, Amino acids within residues 181–200 of the nicotinic acetylcholine receptor alpha 1 subunit in nicotine binding, Biochem. Pharmacol. 55 (1998) 341–347. [12] P.T. Wilson, T.L. Lentz, E. Hawrot, Determination of the primary amino acid sequence specifying the alpha-bungarotoxin binding site on the alpha subunit of the acetylcholine receptor from Torpedo californica, Proc. Natl. Acad. Sci. U. S. A. 82 (1985) 8790–8794. [13] D. Neumann, D. Barchan, M. Fridkint, S. Fuchs, Analysis of ligand binding to the synthetic dodecapeptide of the acetylcholine receptor a subunit, Proc. Natl. Acad. Sci. U. S. A. 83 (1986) 9250–9253. [14] A.H. Taylor, G. Heavner, M. Nedelman, D. Sherris, E. Brunt, D. Knight, J. Ghrayeb, Lipopolysaccharide (LPS) neutralizing peptides reveal a lipid A binding site of LPS binding protein, J. Biol. Chem. 270 (1995) 17934–17938.
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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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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)
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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 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) (⋅-⋅-⋅)
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
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Page 176 and 177: Rho-DOPE (acceptor). The increase o
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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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