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

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Figure I.11 – Experimentally obtained hydrophobicity scales for whole residues (including the peptide bond). Results obtained for the transfer from water to the polar interface of POPC bilayers and from water to octanol are presented (Wimley and White, 1996; Wimley et al., 1996). It is clear from Figure 1.11 that residues like Trp (W), Phe (F), Tyr (Y), Leu (L), Ile (I), Met (M) or Val (V) favour incorporation in hydrocarbon environments, while charged residues are particularly averse to this. From this information and from the primary sequence of a protein it is possible to make previsions for possible alpha helical TM segments inside the protein sequence. A calculation is made through summation of the free energy required to insert (from water into the bilayer hydrocarbon core) successive segments of the polypeptide chain. Segments tested have a finite size, around 20 aminoacids, as this is just about sufficient to spam the hydrocarbon core of a typical bilayer (this aminoacid length corresponds to 30 Å if the helix is oriented along the bilayer normal). This procedure allows the creation of hydropathy plots that signal the polypeptide segments which are the most likely candidates for a TM configuration inside the protein. Aminoacids also exhibit preferential localization in certain positions along the bilayer normal. This fact is of utmost importance to the final position of an alpha-helix in membranes and will be discussed in more detail in section 2 of this chapter. 1.9. Lateral dynamics in biomembranes The Singer-Nicolson fluid mosaic model for biomembranes (1972) (Figure I.12) described the lipid membrane as a two dimensional liquid where both lipids and membrane proteins moved freely. In fact, in pure lipid bilayers in the fluid phase, lipids experience fast lateral diffusion, as well as rotational diffusion, wobbling, and movements in the axis of the bilayer. Crossing from one monolayer to the other is 20
INTRODUCTION: BIOMEMBRANES however much more restricted as discussed in 1.3. Lateral dynamics in the gel phase is approximately 100 times slower (Mouritsen, 2005). The speed of integral membrane proteins is typically 100 times smaller than the speed of lipid molecules. Figure I.12 – The Singer-Nicolson model of fluid mosaic (Taken from Singer and Nicolson, 1972) However, in biomembranes, diffusion of lipids and proteins is hindered by the presence of compartmentalisations of the membrane in the form of domains. Additionally a fraction of the membrane proteins is completely immobile due to interaction with the cytoskeleton network. Generally the average diffusion coefficient of a protein in the plasma membrane of intact cells is about 10-30 times smaller than the one observed in pure lipid liposomes (Mouritsen, 2005). The Singer-Nicolson model is therefore a simplistic description of the complex interactions and dynamics existent in biomembranes. 1.10. Membrane Model Systems Cellular membranes are often too intricate for their properties to be fully resolved as too many variables are present. A valuable alternative is the use of membrane model systems. Model systems grant us a simplified basis for the study of bilayer structure and properties, while at the same time maintaining all the fundamental properties of the biomembrane. Liposomes are the most popular of the membrane model systems. Preparation is often very simple, requiring solubilization of lipids in organic solvents, followed by 21
Page 1: UNIVERSIDADE TÉCNICA DE LISBOA INS
Page 4 and 5: Fernandes, F., Loura, L. M. S., Fed
Page 6 and 7: Ao Pedro e Hugo, amigos de longa da
Page 8 and 9: 2.2. - Peptides as models 2.3. - Am
Page 11 and 12: 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
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Page 54 and 55: amphipatic helices (see Section 2.3
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Page 62 and 63: some α-helical membrane proteins a
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Page 68 and 69: Figure I.20 - Relative binding cons
Page 70 and 71: 2.9. Lipid phase preferential parti
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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
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Page 95: QUANTIFICATION OF PROTEIN-LIPID SEL
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FRET Study of Protein-Lipid Selecti
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BINDING OF INHIBITORS TO A PUTATIVE
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BINDING OF INHIBITORS TO A PUTATIVE
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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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