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

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Figure I.20 – Relative binding constants for OmpF (ο), KscA (∆) and Ca 2+ -ATPase (□) for monounsaturated phosphatidylcholines with different acyl-chains relative to that for DOPC (Taken from Lee, 2003). As already discussed, the amplitude of changes in the relative binding constants of the α-helical proteins, KscA and Ca 2+ -ATPase are not as high as one should expect from equation 2.3 due to adaptation of proteins to more hydrophobically matching configurations, likely by tilting and changes in the packing of α-helices (proteins are not rigid bodies) whereas KscA exhibits preference for lipids with 22 carbons in the acylchain, lipid binding constants for Ca 2+ -ATPase are hardly dependent on acyl-chain, and OmpF is preferentially bound to shorter lipids (14 carbons). Membrane proteins also exhibit selectivity for lipid headgroups. Strong interactions are often found between positively charged TM domains (rich in Lys and Arg) and negatively charged phospholipids. These selectivities can be the result of charge interactions, and in these cases, after binding of the anionic lipid to the surface of the protein, the positive potential in the vicinity of the protein would be slightly reduced. If enough anionic lipid molecules bind to the vicinity of the protein, the positive potential will be neutralized and remaining interactions with anionic phospholipids will be nonspecifical. However, membrane proteins often present different selectivities for different anionic phospholipids. This is likely to result from different balances in charges inside each class of phospholipids. In this way, PS lipids due to the presence of the positively charged ammonium group as well as the negatively charged carboxyl group in the headgroup region may present reduced affinity to basic protein domains when compared to phosphatidic acid. Also, PG, with a single charge in the phosphate 40
INTRODUCTION: LIPID-PROTEIN INTERACTIONS group interacts differently with charged membrane proteins, and proteins often present smaller affinities for PG than for PS and PA (Mall et al., 2002). The complexity to the protein-charged lipid interaction problem is raised by the position of amino acid residues in the protein surface. Basic helices with different hydrophobic thickness were shown to present different selectivities to negatively charged lipids. This was rationalized as the result of tilting of helices in the membrane, that located basic residues away from the headgroup region where strong interactions between proteins and lipid headgroups are more likely (Mall et al., 2002). Additionally, experiments with model peptides showed that insertion of Tyr decreased binding affinities to anionic phospholipids, suggesting that the presence of Tyr residues prevented close association of anionic phospholipids and cationic residues (Mall et al., 2000). These results suggest that the effects of charge on the interactions between lipids and membrane proteins, though important are not determinant, and will be strongly dependent on the detailed structure of the peptide and its orientation in the membrane (Mall et al., 2002). Binding affinities of proteins for lipids are generally presented relative to the binding affinity of PC lipids. PC and PE lipids frequently present the lowest affinities for membrane proteins. Binding sites for hydrophobic molecules have been shown to exist for several proteins distinct from the sites for interaction with annular lipids. These are called nonannular sites. They are generally located between TM α-helices or at protein-protein interfaces. For some cases, these sites are available for interaction with some lipids but not for others (Lee, 2003). Some lipid molecules were shown in crystal structures of proteins to be bound to such sites. These observations suggest that the binding affinities for these sites are much higher than the ones observed for annular lipids (Lee et al., 2003). Notably, several protein domains bind specifically to one or more forms of phosphorylated PIP molecules through interactions that are very specific. Such domains are often found on proteins involved in signal transduction. Other proteins bind specifically to some components of raft domains, establishing a mechanism for targeting the protein to specific structures in the membrane (Epand, 2005; Pérez-Gil et al., 2005). 41
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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
Page 52 and 53: deactivating agents. Zwitterionic d
Page 54 and 55: amphipatic helices (see Section 2.3
Page 56 and 57: size corresponding to the hydrophob
Page 58 and 59: 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 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
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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
Page 114 and 115: 5272 Biochemistry, Vol. 45, No. 16,
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5276 Biochemistry, Vol. 45, No. 16,
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5278 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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