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

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amphipatic helices (see Section 2.3). Predictions derived from these methods allow the design of synthetic peptides for detailed biophysical studies. A number of experiments showed that synthetic peptides corresponding to the alpha-helical TM segments of some proteins can assemble and substitute the native proteins as well as their functions after independent folding, validating the approach of studying isolated peptide segments (Marsh, 1996). Popot and Engelman (1990; 1993) rationalized these results with the proposal of a two-stage model for membrane insertion of the TM segments from a intrinsic membrane protein. In the first stage, alpha-helices are independently folded and inserted in the lipid bilayer, while in the second stage, alpha-helices interact and assemble in the final protein structure. 2.3. Amphipatic helix Amphipatic helices are protein alpha-helices for which one face along the helical axis is hydrophilic whereas the opposite face is hydrophobic. The amphipatic helix is a common feature of biologically active polypeptides and proteins, such as hormones, antibiotics, and venoms. In this way, this structure can play a large number of roles. A function of great structural importance is the shielding of the hydrophobic interior of proteins exposed to aqueous environments, while for membrane proteins the roles played by amphipatic helices are diverse. In the case of peripheric proteins they can act as anchors to the membrane by tight binding to the hydrocarbon/water interface of the bilayer as the structure of this boundary is highly complementary to the amphipatic helix. The amphipatic structure is also able to promote protein-protein interactions between hydrophobic segments of helices. In some cases this allows amphipatic peptides to create channels and pores in the membrane via aggregation into oligomeric bundles for which the hydrophobic side faces the hydrocarbon chains of the lipids and the hydrophilic faces of the helices are exposed to the inside. Thus delineating a pore in the bilayer (Epand, 1993). Recent studies also suggest a role of amphipatic helices in the modulation of membrane curvature (see chapter V). Overall, the amphiphilic character is a much more important dictator of peptide conformation than the specific characteristic of its constituent amino acids (Taylor, 1990). Parameters like the ratio of hydrophobic to hydrophilic amino acids as well as 26
INTRODUCTION: LIPID-PROTEIN INTERACTIONS the angle of the hydrophobic face are crucially important in dictating the location of the peptide in the membrane and the type of lipid interactions established (Kitamura et al., 1999; Brasseur, 1991). Methods exist to predict possible amphipatic helices from primary sequences of proteins. From the knowledge of the periodicity of alpha-helices, Shiffer-Edmundson’s (1967) helical wheels and helical net representations (Dunnil, 1968) can be drawn like the ones in Figure I.13. In Shiffer-Edmundson’s representation, residues are sketched around a circle with each residue being placed 100º from the preceding one. If the segment corresponds to an amphipatic helix, then one face of the circle should be enriched in hydrophilic residues while the opposite face should present clustering of hydrophobic amino acids. The net helical representation illustrates a hollow cylinder cut open along a parallel line to its axis and laid flat. The residues are placed in such a way so that if the cylinder would be closed, the amino acids are positioned as in a alphahelix (Auger, 1993). These methods are very easy to apply and provide great tools to visualize the disposition of the amino acids in a putative amphipatic helix but are not quantitative, i.e. they do not provide a quantification of the amphipatic character of the protein domain being evaluated. A B Figure I.13 - A- Shiffer-Edmundson’s helical wheel representation for amphipatic domains. B- Helical net representation for amphipatic domains (Taken from Anantharamaiah et al., 1993). Some of the available quantitative methods work by comparing a sequence of hydrophobicities (of each amino acid) with a sinusoid, reporting the evolution of the amino acid positions in the alpha-helical polypeptide chain (in the axis perpendicular to the helical axis). The most common quantitative description of the amphipatic degree of a protein domain is given by the hydrophobic moment (Eisenberg et al., 1982). In this analysis, the hydrophobic moment of a helix is equal to the summation of the vectors of 27
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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 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 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 and 78: PROTEIN-PROTEIN AND PROTEIN-LIPID I
Page 79: 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
Page 93 and 94: 2440 Fernandes et al. tein oligomer
Page 95: QUANTIFICATION OF PROTEIN-LIPID SEL
Page 98 and 99: FRET Study of Protein-Lipid Selecti
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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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