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

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particle is 900 nm in length and 6.5 nm wide. It is formed by a single strand of DNA, about 6400 nucleotides long, encapsulated by a cylindrical protein coat. The protein coat presents about 3000 copies of the major coat protein (mcp or gp8), the product of gene VIII. In addition to mcp, four other coat proteins are present in few copies, capping the ends of the bacteriophage. During the infection process, parental coat proteins are embedded in the membrane while the viral DNA is released in the cytoplasm (see Figure II-1). New copies of mcp are synthesized in the host and inserted in the membrane, where they are processed by a leader peptidase (Kuhn et al., 1986). After peptidase action, the C-terminus of mcp is oriented to the cytoplasm and the N-terminus is located in the periplasm. In the mature form, mcp is a polypeptide chain 50 aminoacids long and presents a single transmembrane domain. During the reproductive cycle of the bacteriophage, coat proteins are stored at very high local levels and finally assembled around viral DNA into new phage particles (Hemminga et al., 1992). Figure II-1: Representation of the reproductive cycle of bacteriophage M13 (taken from Spruijt et al., 1999). mcp presents three distinct domains which are expected to be related to the distinct type of interactions that this protein is involved in during the reproductive cycle of the bacteriophage. In this way, the negatively charged region in the N-terminal domain of the protein (residues 1-20), rich in glutamate and aspartate, forms the outer hydrophilic surface of the virus particle. The hydrophobic central domain (residues 21-39) is responsible for the tight membrane interaction of the membrane form of mcp and for the 48
PROTEIN-PROTEIN AND PROTEIN-LIPID INTERACTIONS OF M13 MCP protein-protein interactions between mcp units inside the phage coat, which are essential in providing stability to the particle. Finally, the C-terminus of mcp (residues 40-50) is enriched in positively charged lysines. This domain lines the inside of the phage particle and is responsible by non-specific interactions with DNA. The amino acid sequence of mcp and the structure of the membrane bound form is shown in Figure II-2. The N-terminus of the membrane bound form of mcp is a flexible α-helix with amphipatic character. A loop connects this amphipatic domain with the transmembrane helix (Figure II-2). The transmembrane domain of mcp is tilted by 20 ± 10º with respect to the lipid bilayer normal (Glaubitz et al., 2000; Koehorst et al., 2004). ESR and fluorescence studies making use of site-directed labelling of mcp (Spruijt et al., 1996; Stopar et al., 1997a) allowed to conclude that Thr 36 is located in the center of the bilayer in DOPC and 1,2-dioleoyl-sn-glycerol-3-phosphatidylglycerol (DOPG) bilayers, while aminoacids 25 and 46 delineate the edges of the transmembrane domain of mcp. DOPC is expected to present ideal hydrophobic matching conditions to mcp. A B Figure II-2: A - Amino acid sequence of mcp (taken from Hemminga et al., 1992). B – Structure of the membrane bound form of mcp (taken from Stopar et al., 2003). mcp was also found to be strongly anchored to the lipid bilayer through its C- terminus, namely by the two phenylalanines found there and by three lysine residues. When mcp was incorporated in bilayers with slightly thinner or thicker hydrophobic 49
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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
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
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: PROTEIN-PROTEIN AND PROTEIN-LIPID I
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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
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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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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