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

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The formation of a lipid population associated with proteins or peptides with properties different from the bulk or free lipid was predicted from results of different techniques, namely differential scanning calorimetry and electron spin resonance (ESR). From ESR experiments, a fraction of the spin labelled lipids was found to be dynamically restricted by interaction with membrane proteins or peptides. From the number of lipid molecules affected by the presence of a single TM domain it was possible to retrieve the stoichiometry for the interaction of lipids and TM peptides. The value recovered was 12, meaning that only the first shell of lipids around the TM domain is significantly affected by its presence (a hexagonal-type arrangement of lipids around a TM peptide is expected, six in each monolayer – see Figure I.17) (Marsh et al., 2002). This shell around the TM domain is entitled the annulus and the lipids within are called annular lipids. Figure I.17 – Hexagonal lipid packing of annular lipids around a TM protein domain. Annular lipids are shown in dark grey and bulk lipids are depicted in light grey. The fact that only the first shell of lipids around the TM domain is significantly affected explains why single TM peptides are generally not able to induce considerable physical changes in the bulk membrane (except when the lipid/peptide ratio is large enough so that a significant fraction of the lipids are annular lipids). However, multipass TM proteins (containing multiple TM domains) were already shown to be able to affect the bulk lipids and change the hydrophobic thickness of the bilayer (Harroun et al., 1999). These results seem to indicate that the packing of lipid chains around a single α- helix (which has a diameter ~ 10 Å, comparable to a lipid molecule) is significantly different from the way lipids pack against a large protein surface (Weiss et al., 2003). Still, when multipass TM proteins were studied by ESR with spin labelled-lipids, the ratio of immobilized lipids/TM domains is often no longer 12 but smaller. This is obviously due to interactions and contacts between the TM domains that decrease the area available for contact with lipids (Marsh et al., 2002). However, if lipids outside the 36
INTRODUCTION: LIPID-PROTEIN INTERACTIONS annulus were considerably restricted by the presence of protein, the immobilized lipids/TM domains ratio should come larger that for a single α-helix. Likely, the influence of the TM proteins on the annular lipids is much stronger than on lipids on the exterior shells, and the effects on the latter are not detectable by ESR. The lipid exchange rate at the interface of the protein body was determined to be in the region of 10 7 s -1 , which is on the same order but significantly lower than the value obtained for lipid diffusion in the absence of protein (Marsh, 1990). This means that although temporarily restricted to the protein surface, the lipid molecules interacting with the protein body are not immobile and are still in exchange with other lipids. Of relevance is the fact that lipids display different properties when at the surface of proteins. This indicates that optimal hydrophobic matching might not necessarily occur when the hydrophobic thickness of the protein matches the hydrophobic thickness of the unperturbed bilayer (Dumas et al., 1999). 2.8. Lipid selectivity at protein interfaces For membranes containing multiple lipid species, the hydrophobic matching principle can result in even more complex behaviour at the lipid-protein interface. Different lipid species are expected to present different free energies for interaction with the presented TM domain and as a result, the lipid composition around the protein can be different from the bulk lipid composition (Figure I.18). Selectivity of membrane proteins for lipids in specific phases was observed in various cases. In the presence of a mixture of DLPC and DSPC, a combination of experimental and theoretical approaches indicated that bacteriorhodopsin exhibited preferential interaction with DLPC at low temperatures, when both lipids where in the gel phase, and at the fluid phase, bacteriorhodopsin had a preference for long chain DSPC (Dumas et al., 1997). DLPC and DSPC present a very large difference in hydrophobic length of acyl-chains and strongly nonideal mixing. Consequently, it is not surprising that the affinity of the protein for the lipids differs drastically, and that the protein is preferentially solvated by the lipids that are the best hydrophobic match in each phase. Still, selectivity of proteins for lipids in binary mixtures with a much more ideal mixing is also experimentally verified (Lee, 2003). Nevertheless, deviation of a 37
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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
Page 13: 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 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
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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
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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) (⋅-⋅-⋅)
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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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