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

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L o L/P LUV MARCKS mcp MLV n S,i PI(4)P PI(5)P PI(4,5)P 2 PI(3,4,5)P 3 PL i P β’ PA PC PE PG PI PS R SDS sn s o SUV V i T TFE T m TM T p W Liquid-ordered phase Lipid to protein/peptide ratio Large unilamellar vesicle Myristoylated alanine-rich C kinase substrate Major coat protein Multilamellar vesicle Moles of solute in phase i Phosphatidylinositol-(4)-phosphate Phosphatidylinositol-(5)-phosphate Phosphatidylinositol-(4,5)-bisphosphate Phosphatidylinositol-(3,4,5)-trisphosphate Complex of protein and lipid species i Rippled Gel or Periodic Gel Phase Phosphatidic acid Phosphatidylcholine Phosphatidylethanolamine Phosphatidylglycerol Phosphatidylinositol Phosphatidylserine Ideal gas constant Sodium dodecyl sulfate Stereospecific number Gel or solid-ordered phase Small unilamellar vesicle Volume of phase i Temperature Trifluoroethanol Main transition temperature Transmembrane Pre-transition temperature Aqueous phase
RESUMO RESUMO As biomembranas são responsáveis pela operação de diversas funções celulares e a multiplicidade destas interacções requer uma composição química complexa. Apesar da estrutura básica das biomembranas ser a bicamada lipídica, outros componentes estão presentes, em particular proteínas de membrana. Estes diferentes componentes apresentam energias de interacção distintas, induzindo frequentemente heterogenidades na distribuição lateral de biomembranas. Neste trabalho foram estudadas as interacções entre componentes de biomembranas em diferentes sistemas. É demonstrado que a distribuição lateral da proteína principal da cápside do bacteriófago M13 (mcp) é dependente da composição lipídica da membrana. Uma nova metodologia para quantificação de selectividades proteína-lípido foi desenvolvida e aplicada com sucesso à mcp. Estudos de ligação entre drogas e proteínas de membrana foram conduzidos seguindo diferentes aproximações. No estudo da ligação de inibidores de V-ATPase a um domínio transmembranar do enzima, uma aproximação reducionista foi escolhida, enquanto que no estudo da ligação do ciprofloxacin para a OmpF, a proteína foi utilizada na forma intacta. A interacção entre um segmento N-terminal anfipático de um domínio BAR com membranas foi investigada e conclusões foram obtidas relativamente ao papel deste segmento na remodelação de membranas por domínios BAR. Finalmente, a hipótese de agregação espontânea de fosfoinositol-(4,5)-bisfosfato foi testada e excluída. PALAVRAS‐CHAVE Interacções Proteína‐Lípido Proteína Principal da Cápside do M13 Inibição do V‐ATPase PI(4,5)P 2 Domínios BAR FRET xi
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: ABBREVIATIONS AND SYMBOL LIST ABBRE
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
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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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2430 Biophysical Journal Volume 85
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2432 Fernandes et al. Coat protein
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2434 Fernandes et al. leads to an a
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2436 Fernandes et al. FIGURE 4 (A)
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2438 Fernandes et al. section of th
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2440 Fernandes et al. tein oligomer
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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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