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

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interact differentially, depending on the distribution of polar and hydrophobic residues on the protein and on the size of the hydrophobic lipid domain. These changes in selectivities of association can lead to changes in the lateral distribution of the membrane components in a similar manner as observed for lipid mixtures. In this way, a heterogeneous distribution of lipids can drive membrane protein heterogeneity and vice-versa. In fact, it is known that proteins and lipids are often distributed in a heterogeneous manner in the membrane, and this is essential in ensuring the localized character of some events in cellular membranes. Drugs are another type of agents which often display affinity for lipid membranes. The targets of several drugs are membrane proteins, and their action is frequently expected to be exerted in the membrane environment. Therefore, interaction of drugs with biomembrane components is of great biological relevance. In addition, for drugs targeting proteins inside the cell, partition to the plasma membrane is the first step to cell entry, and water/lipid partition coefficients are in this way, relevant parameters in drug research. It is clear that interactions between biomembrane components are used by cells to mediate and localize diverse functions in the cell membrane. A better understanding of these mechanisms is the first step in rationalizing biomembranes and their functions. However, cellular membranes are often too intricate for the properties of some membrane component interactions to be resolved, and a valuable alternative is the use of membrane model systems. Here, the majority of the studies were carried out in large unilamellar vesicles, with a diameter around 100 nm. This model system granted us a simplified basis for the study of the interactions between membrane components, at the same time maintaining all the fundamental properties of the biomembrane. Fluorescence techniques are ideal tools to study lateral distribution of membranes. By choosing an intrinsic fluorescent amino acid (Trp or Tyr), or fluorescent labelling of proteins and lipids, we can monitor fluorescence changes (intensity, lifetime, anisotropy) upon interaction with other membrane components. Fluorescence quenching studies can give information on the collision efficiency, and this can give us indications on: i) the location of the fluorescent group; ii) on the concentration of quencher; and iii) on the diffusion properties of both quencher and fluorescent molecule. One technique that was particularly useful in evaluating lateral distributions in the membrane was Förster resonance energy transfer (FRET). FRET efficiency for a single donor-acceptor pair is strongly dependent on the distance between the two. In the case of lipid
OUTLINE membranes with a distribution of donors and acceptors, FRET will be sensitive to changes in the distribution of acceptors around the donor population in the space range of the Förster radius (up to ~10 nm depending on the donor-acceptor pair). Several different systems were investigated. In Chapter II, an integral protein from the coat of the bacteriophage M13 (mcp) was studied in model membranes. The oligomerization state of the protein changes repeatedly during the reproductive cycle of bacteriophage M13, but the state of the protein in the membrane was thought to be monomeric as this is apparently required for correct insertion in nascent bacteriophages. However, this was still matter of debate, as in the past, oligomeric mcp was shown to exist in some conditions. Here, it is shown that mcp is a monomer when incorporated in membranes composed by lipids with hydrophobic thickness that matched the hydrophobic thickness of the protein (monounsatured chains C 18 chains). On the other hand, when bilayers with thicker hydrophobic thickness (monounsatured chains C 22 chains) were used, aggregation was observed. Surprisingly, when mixtures of both matching and mismatching lipids were used, segregation of mcp to domains enriched in matching lipid was apparently induced, and while in these domains, mcp remained monomeric. This result is a dramatic demonstration of the relevance of hydrophobic matching for protein-lipid organization. Only a small increase of 4 carbons in the acylchain of the matrix lipid can drive protein aggregation, and in case that a significant fraction of matching lipid is present, lipid-lipid segregation can be induced by the presence of the protein. A FRET methodology was developed to quantify the degree of selectivity exhibited by mcp to different lipids. The results obtained for lipids with different headgroups are almost perfectly matched to previous results obtained with electron spin resonance (ESR) for the same protein, even though in an aggregated state. This agreement establishes the FRET methodology for quantification of protein-lipid selectivity described here as an alternative to the use of ESR. This FRET methodology was also applied to the study of selectivity for acyl-chain length, which is as already mentioned, essential in dictating the lateral distribution of the protein in liposomes. Also surprisingly, the results revealed weak selectivity constants for a hydrophobically matching lipid, both in thinner as in thicker bilayers. Drug binding to membrane proteins was studied following two different strategies. In chapter III, a reductionist approach was used. A peptide expected to correspond to the putative 4 th transmembrane domain of the membrane bound c-subunit of V-ATPase, xxiii
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: OUTLINE OUTLINE The last two decade
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
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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
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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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