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

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thickness than mcp, the N-terminus of the protein shifted its position in order to accommodate the differences in hydrophobic lengths of protein and lipid, but the C- terminus remains at approximately the same position in the bilayer interface due to strong anchoring (Meijer et al., 2001). Another adaptation of mcp to hydrophobic mismatch is changing the tilt angle of the transmembrane domain, as well as the rotation of the helix at its long axis so as to optimize the hydrophobic and electrostatic interactions at the C-terminus (Koehorst et al., 2004). Several questions concerning lipid-protein and protein-protein interactions of mcp are still subject of debate. During the life-cycle of bacteriophage M13, the aggregation state of mcp changes repeatedly. In the phage particle it is aggregated in the coat, and in the membrane it is believed to be monomeric (Russel, 1991; Spruijt and Hemminga, 1991), although a structural dimer might be formed as an intermediate species during phage disruption (Henry and Sykes, 1992; Stopar et al., 1997b; Stopar et al., 1998). The mcp structure is predominantly α-helical when inserted in the membrane, but depending on the protein purification and membrane reconstitution procedure a irreversibly aggregated β-sheet conformation of the protein appeared (Hemminga et al., 1992). Also, factors such as L/P ratio, phospholipid headgroup type, length and degree of unsaturation of the acyl-chains of lipids used, and the ionic strength of the buffer were shown to be able to influence the α-helical to β-sheet transition. It is found that saturated lipid chains and very high protein concentration resulted inevitably in high levels of irreversible β-sheet aggregation (Spruijt and Hemminga, 1991). Nevertheless, the irreversible β-sheet conformation of mcp is expected to be an artefact resulting from inappropriate conditions of protein purification and reconstitution, as α-helical structure is necessary for insertion in the phage particle (Hemminga et al., 1992). The lipid composition of the inner membrane of non infected E. coli is about 70% of PE, 25% of PG and 5% cardiolipin. During the infection of E. coli by the M13 bacteriophage, the levels of anionic lipids in the cell membrane are slightly increased and blocking of phage assembly resulted in significant increases in anionic lipid content in the inner membrane of bacteria (Pluschke et al., 1978). This phenomenon might indicate that anionic lipids are required to maintain a functional state of the membranebound form of the protein (Hemminga et al., 1992). The in situ aggregational state of α-helical mcp is however still a matter of discussion, as reversible oligomeric alpha-helical mcp was shown to exist in some conditions (Hemminga et al., 1992). In vitro, aggregation behaviour is even more likely 50
PROTEIN-PROTEIN AND PROTEIN-LIPID INTERACTIONS OF M13 MCP than in vivo, as the reconstitution procedures normally result in randomized orientations for mcp instead of the single orientation observed in vivo. mcp in the membrane with identical orientations are expected to present smaller protein-protein electrostatic attractive forces due to the stronger repulsion between the heavily charged C-terminus, and consequently a higher free energy is expected for the oligomeric condition. In order to achieve high local concentrations of mcp in the host membrane prior to phage assembly, a dynamic protein-lipid network should be present, characterized by absence of preferential association between coat proteins and/or lipids (Hemminga et al., 1992). Concerning the protein-lipid interactions established by mcp, it is interesting to note that the monomeric form of mcp was not able to induce sufficient immobilization of a fraction of lipid molecules so that it could be detected by ESR with spin labelled lipids (Sanders et al., 1992). On the other hand, oligomeric mcp immobilized a population of lipids, especially at very high protein concentrations (Peelen et al., 1992). Thus, it can be concluded that monomeric M13 mcp is not able to induce a long living lipid boundary shell around it. Possible explanations are that the hydrophobic surface of mcp exposed to the lipids is too small or that the diffusion rate of the protein is not sufficiently lower than that of the lipids. 51
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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
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SINOPSE SINOPSE Nas últimas duas d
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SINOPSE podem fornecer informação
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SINOPSE aceitantes. Dadores mais pr
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OUTLINE OUTLINE The last two decade
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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 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: 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
Page 114 and 115: 5272 Biochemistry, Vol. 45, No. 16,
Page 123: BINDING ASSAYS OF INHIBITORS TOWARD
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1780 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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