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

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homogeneous distribution of lipids in the membrane does have an energetic entropic cost and the degree of protein-lipid selectivity is a balance between the need to satisfy hydrophobic matching conditions in the protein-lipid interface and the intrinsic tendency of the system for mixing. Figure I.18 – Illustration of a lipid bilayer with an embedded protein and two different lipid species. The hydrophobic matching principle implies an accumulation of the lipid species that is hydrophobically best matched to the protein (taken from Jensen and Mouritsen, 2004). The hydrophobic surface of a membrane protein is not smooth. The interface between the protein and the lipids surrounding it is likely to be heterogeneous, and the interactions taking place there complex (Lee, 2003). Still the results described above, concerning the presence of a fixed stoichiometry for annular lipids, denote some degree of ordering in the TM alpha helix-lipid interface and in this way, processes of lipid binding to simpler systems such as single TM domains are suitable to be described in terms of a uniform surface for which several (12) identical binding sites are available (Marsh et al., 2002). With total coverage of the protein surface by lipids, one lipid molecule must leave the surface before another can enter. In this sense, the process can be depicted as competitive binding of lipids at the binding sites on the protein surface (Lee, 2003) (Figure I.19). This method of analysis has been applied with success to even more complex membrane proteins (O’ Keefe et al., 2000; Williamson et al., 2002; Powl et al., 2003). 38
INTRODUCTION: LIPID-PROTEIN INTERACTIONS Figure I.19 – View from above of lipid binding sites on a TM domain surface. Two lipid species (L 1 and L 2 ) are shown exchanging at one site. At each site an equilibrium exists: PL 1 + L 2 PL 2 + L 1 where PL 1 and PL 2 are the protein-lipid complexes with lipid species 1 and 2. This equilibrium can be described by a binding constant (K b ): b [ PL 2 ] ⋅[ L1] [ PL ] ⋅[ L ] K = 2.4 1 2 If L 2 is present at small amounts in the system (L 2 > [PL 2 ], then the probability (µ) of a lipid annular site to be occupied by L 2 is: [ PL 2 ] [ PL ] + [ PL ] 2 1 [ PL 2 ] [ PL ] 1 b [ L 2 ] [ L ] µ = = = K ⋅ 2.5 1 Through knowledge of [PL 2 ] ([PL 1 ]=[P]-[PL 2 ]), and the concentration of the lipid species 1 and 2 it is then possible to recover a binding constant for protein-lipid selectivity. The most popular methodologies for quantifying protein-lipid selectivity have been the already described ESR measurements with spin labelled lipids, and measurements of protein fluorescence quenching by brominated lipids (East and Lee, 1982; O’ Keeffe et al., 2000; Williamson et al., 2002; Powl et al., 2003). From these studies, binding constants were retrieved as function of fatty-acid chain for a series of proteins (Fig. I.20). 39
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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
Page 17 and 18: SINOPSE SINOPSE Nas últimas duas d
Page 19 and 20: SINOPSE podem fornecer informação
Page 21 and 22: SINOPSE aceitantes. Dadores mais pr
Page 23 and 24: OUTLINE OUTLINE The last two decade
Page 25 and 26: OUTLINE membranes with a distributi
Page 27: OUTLINE BAR domains (tubulation of
Page 30 and 31: ion diffusion, as the energy requir
Page 32 and 33: acid. If no more groups are linked
Page 34 and 35: sphingomyelin, the most abundant sp
Page 36 and 37: signal for neighbouring cells to ph
Page 38 and 39: functional role in process such as
Page 40 and 41: in the L α phase, while lateral di
Page 42 and 43: 1.7. Lateral heterogeneity in lipid
Page 44 and 45: the vesicle through bilayer deforma
Page 46 and 47: Figure I.9 - Depiction of the sever
Page 48 and 49: Figure I.11 - Experimentally obtain
Page 50 and 51: drying into a film and ressuspensio
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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
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Page 75 and 76: PROTEIN-PROTEIN AND PROTEIN-LIPID I
Page 77 and 78: 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)
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,
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5274 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) (⋅-⋅-⋅)
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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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