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

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CONCLUSIONS VII CONCLUSIONS Chapter II ‐ Protein‐Protein and Protein‐Lipid Interactions of M13 Major Coat. In this chapter an extensive study of the protein-protein and protein-lipid interactions of M13 mcp was conducted. The fluorescence self-quenching data obtained from the study with a BODIPY labelled mcp allowed us to conclude that mcp is in fact monomeric in DOPC bilayers at the conditions used for protein purification and protein reconstitution. The use of two different sites for labelling, resulted in identical results excluding any possible influence of labelling on the oligomerization efficiency of mcp. Nevertheless, as the studies with lipid bilayers presenting a different hydrophobic thickness than mcp shows, mcp can aggregate is certain conditions. In this way, it is possible that at much higher protein concentrations than the one used here (L/P < 50), some aggregation takes place, although in vivo the presence of only one orientation of mcp should contribute to keep it monomeric. This could explain the increase in the levels of anionic lipids (cardiolipin and phosphatidylglycerol) in the lipid membrane of E. coli during the process of infection by the bacteriophage M13. Therefore, although anionic lipids are not required to keep mcp monomeric in normal conditions, at the very high mcp concentrations found in the sites of phage assembly in the lipid membrane of the host, they might contribute to stabilize the monomeric state of the protein, which is required for correct assembly. Fluorescence self-quenching of BODIPY labelled mcp is more efficient when mcp is incorporated in bilayers with a thinner (positive hydrophobic mismatch) or thicker (negative hydrophobic mismatch) hydrophobic length, than the hydrophobic domain of mcp. This is likely to result from aggregation of mcp, and in this case aggregation in thicker bilayers is significantly more effective than in thinner bilayers. This discrepancy can be the result of the availability of alternative methods for protein adaptation in positive hydrophobic mismatch conditions. In this case, the protein is able to increase 181
the tilt angle, and maintain a larger section of the hydrophobic domain inside the bilayer (Figure I.16). When the bilayer thickness is larger than the protein hydrophobic domain, the TM protein might find that compensation by change of tilt angle is not possible or it is insufficient, and the only route available to decrease exposure of hydrophobic aminoacid side-chains is aggregation. Recently, mcp was shown to change its tilt angle when incorporated in bilayers with different hydrophobic thickness (Koehorst et al., 2004). Results obtained for fluorescence self-quenching of BODIPY labelled mcp incorporated in binary lipid mixtures composed by lipids with different acyl-chain lengths are dramatically different from the results obtained with pure lipids. The apparent diffusion coefficients retrieved from the analysis of this data are clearly not justified on the basis of normal diffusion of monomeric species (as it was for mcp in DOPC), or on the basis of protein oligomerization/aggregation (as it as for mcp in DMoPC and in DEuPC). In this case, only segregation of mcp into domains enriched in the hydrophobically matching lipid can provide rationalization of the data. This hypothesis is confirmed by FRET studies that are also consistent with mcp segregation in the presence of binary mixtures of lipids with different acyl-chains. Importantly, the lipid components used in the binary mixtures are not dramatically different, the only difference being the presence of 4 additional carbons in the acyl-chain of one of the lipids. This difference is not expected to result in large scale phase separation of the lipid components such as it was detected in the FRET studies (domain size larger than or comparable to the Förster radius of AEDANS-BODIPY, 48.8 Å). In this way, formation of large domains enriched in the hydrophobically matching lipid must be a consequence of incorporation of mcp. Nevertheless, experiments making use of the properties of 1,6-diphenylhexatriene, indicate that even in the absence of protein, short scale clustering of the lipids in the binary mixtures is expected to occur. The same type of segregation of mcp was not detected when the acyl-chains’ lengths of the lipid components in the binary mixtures were identical and the only difference resides in the headgroups, confirming the crucial relevance of hydrophobic matching for protein organization. Concerning the quantification of the lipid selectivity of mcp, FRET proved to be a valuable tool, and an important alternative to ESR in these type of studies. FRET was able to detect lipid enrichment around monomeric mcp in a binary lipid mixture, while ESR was unable to resolve this problem (Sanders et al., 1992). Relative association 182
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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
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OUTLINE BAR domains (tubulation of
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ion diffusion, as the energy requir
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acid. If no more groups are linked
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sphingomyelin, the most abundant sp
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signal for neighbouring cells to ph
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functional role in process such as
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in the L α phase, while lateral di
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1.7. Lateral heterogeneity in lipid
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the vesicle through bilayer deforma
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Figure I.9 - Depiction of the sever
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Figure I.11 - Experimentally obtain
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drying into a film and ressuspensio
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deactivating agents. Zwitterionic d
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amphipatic helices (see Section 2.3
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size corresponding to the hydrophob
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changes abruptly in the interfacial
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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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Page 166 and 167: dynamin. Soon after, the same group
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Page 172 and 173: Introduction Control of membrane re
Page 174 and 175: Steady-state fluorescence measureme
Page 176 and 177: Rho-DOPE (acceptor). The increase o
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Page 182 and 183: 19. Fernandes, F., L. M. S. Loura,
Page 184 and 185: Figure Legends Fig.1: N-terminal am
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Page 188 and 189: FIG. 3A FIG. 3B 19
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Page 196 and 197: The signalling functions of PI(4,5)
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Page 202 and 203: zoxadiazol (NBD)-PC were obtained f
Page 204 and 205: Fig. 3. Fluorescence decays of diph
Page 208 and 209: CONCLUSIONS constants recovered by
Page 210 and 211: CONCLUSIONS Chapter IV ‐ Binding
Page 212 and 213: CONCLUSIONS Chapter VI - Clustering
Page 214 and 215: FINAL CONSIDERATIONS AND PROSPECTS
Page 216 and 217: BIBLIOGRAPHY IX BIBLIOGRAPHY Albert
Page 218 and 219: BIBLIOGRAPHY Phosphatidylinositol 4
Page 220 and 221: BIBLIOGRAPHY Glaubitz, C., Grobner,
Page 222 and 223: BIBLIOGRAPHY Koehorst, R. B. M., Sp
Page 224 and 225: BIBLIOGRAPHY Mishra, V. K., Palguna
Page 226 and 227: BIBLIOGRAPHY Pluschke, G., Hirota,
Page 228 and 229: BIBLIOGRAPHY Sperotto, M. M., and M
Page 230: BIBLIOGRAPHY Williamson, I. M., Alv
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