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

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Rho-DOPE (acceptor). The increase of FRET efficiencies after the mixture reports fusion of the liposomes. H0-NBAR stimulated fusion for both POPG (considerably) and POPC liposomes (slightly) (Fig. 4) and was more effective in doing so than a control peptide (H0- ENTH), corresponding to the helix 0 of the epsin N-terminal homology domain that is also related to membrane curvature induction (albeit in the case of epsin the presence of phosphatidylinositol-(4,5)-bisphosphate PIP(4,5) 2 is required for membrane remodeling) [20]. It was checked that under these conditions H0-ENTH is completely bound to liposomes (results not shown). H0-NBAR is an antiparallel dimer in the membrane The function of H0-NBAR in liposome tubulation mediated by the BAR domain was recently proposed to be related to BAR domain oligomerization (7). With the intent to verify if H0-NBAR oligomerizes in the membrane, FRET measurements were performed using H0-NBAR-EDANS as a donor and H0-NBAR-FITC as an acceptor (emission and absorption spectra in Supplementary Data). For this system, the high Förster radius (R 0 ) of the EDANS-FITC donor-acceptor pair (R 0 (EDANS-FITC) = 40 Å) entails a small contribution of energy transfer between nonoligomerized H0-NBAR and this was taken into account in our analysis (see FRET simulation for the monomeric hypothesis in Fig.5A). The donor fluorescence decay in the presence of acceptors is described as, n−1 nk - i () t = ⎡ f ( k)ρ () t ⎤ ()ρ DA ⎢∑ ⋅ ⋅i t ⋅ Dq nonbound () t + bound D ⎣ ⎥ k= 1 ⎦ n−1 ⎛ 1 f ( k) ⎞ ⎜ −∑ i ( t) ρ t Dq ⎟⋅ ⋅ D nonbound ⎝ k= 1 ⎠ () [3] where ρ bound is the FRET contribution from energy transfer to each acceptor in the oligomer containing the donor, ρ nonbound is the FRET contribution arising from energy transfer to acceptors that are not integrated in the same oligomer as the donor, and f Dq (k) is the fraction of donors bound to k acceptors, Here n is the number of units in the oligomer, k counts the number of donors, P D is the mole ratio of donors and P A is the mole ratio of acceptors. Through measurement of the dependence of FRET efficiencies on the acceptor/donor ratio it is possible to conclude about the presence of oligomerization and to gather information on the type of oligomerization found (21). The contribution for FRET from each acceptor in the same oligomer as the donor is given by, ρ n ( k ) D ( ) k n k fDq k = k⋅ ⋅P P ⎛ 6 1 ⎛ R ⎞ 0 bound = exp − ⎜ ⋅⎜ ⎟ τ0 rD-A ⎝ − A ⎝ ⎞ ⎠ ⎟ ⎠ [4] [5] 7
where τ 0 is the lifetime of the donor in the absence of acceptors, and r D-A is the distance between the donor and the acceptor inside the oligomer. Equation 6 gives ρ nonbound for a two dimensional system (15). nonbound 2 [ ρ = exp −n ⋅π ⋅Γ(2 / 3) ⋅ R 2 0 ⎛ t ⎞ ⋅⎜ ⎟ ⎝τ 0 ⎠ 1 3 ] [6] where n 2 is the acceptor density in each bilayer leaflet, and Γ is the gamma function. From Eqs. 2-6, simulations for FRET efficiencies are obtained (Fig. 5A). In the FRET analysis, the different oligomerization models were fitted to two series of data simultaneously. In the first series the lipid to protein ratio (L/P) equals 1000 while in the second the protein was diluted two fold (L/P = 2000). With this methodology, uncertainty resulting from erroneous quantification of the two FRET contributions (to acceptors within the same oligomers and to unbound acceptors) is eliminated. The oligomerization model that provided the best fit to the two data series was one that considered dimerization of the peptide and a distance of 43 Å between donor and acceptor inside the dimer. This distance is in good agreement with an antiparallel dimer as the size of a rigid and fully alpha-helical H0-NBAR is expected to be around 49.5 Å (1.5 Å per residue). The sensitivity of the methodology chosen is clear from the comparison with the simulations for FRET efficiencies arising from a parallel dimer (r D-A < 20 Å) and other oligomerization numbers (see Fig. 5A). The oligomerization of H0-NBAR is dependent on membrane binding, as the peptide exists as a monomer while in buffer. This was concluded from anisotropy decays of H0- NBAR-FITC (Supplementary Data). The anisotropy decays were well described by two rotational correlation times, φ 1 and φ 2 . rt () ⎡ ⎛−t ⎞ r ⋅ ⎢ β ⋅ exp⎜ ⎟+ ⎣ 0 1 = ⎝ φ1 ⎠ ⎛−t ⎞⎤ β2 ⋅exp⎜ ⎟⎥ ⎝φ2 ⎠⎦ [7] Here r 0 is the fundamental anisotropy, β 1 and β 2 are the component amplitudes. The methodology for analysis of time-resolved anisotropy decays is described in detail elsewhere (16). φ 1 was typically smaller than 250 ps and is expected to be related to independent and fast movement of the FITC fluorophore. The recovered φ 2 values were between 1,45 and 1,69 ns (Fig. 5B) and are expected to correspond to the motion of the entire peptide. The rotational correlation time can be estimated from the following equation (strictly valid for spherical molecules), η ⋅V φ= R ⋅T [8] 8
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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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Page 140 and 141: Introduction Quinolones are broad-s
Page 142 and 143: [9-12]. Having this into considerat
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Page 146 and 147: placement of the protein around the
Page 148 and 149: ⎛ II t ⎛ R ⎞ 0 ρ () t = exp
Page 150 and 151: APPENDIX - Derivation of the FRET r
Page 152 and 153: 2 2w J ( t) = '2 R − R + w / 1
Page 154 and 155: [14] L. Plançon, M. Chami, L. Lete
Page 156 and 157: acceptors) (Eqs. 4-6) (⋅-⋅-⋅)
Page 158 and 159: FIGURE 1A FIGURE 1B 130
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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 178 and 179: where η is the viscosity of the me
Page 180 and 181: ound conformation of the BAR domain
Page 182 and 183: 19. Fernandes, F., L. M. S. Loura,
Page 184 and 185: Figure Legends Fig.1: N-terminal am
Page 186 and 187: FIG. 1A FIG.1B 17
Page 188 and 189: FIG. 3A FIG. 3B 19
Page 190 and 191: FIG.5A 21
Page 192 and 193: FIG 6. 23
Page 194 and 195: FIG. 8 A B 25
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 206 and 207: CONCLUSIONS VII CONCLUSIONS Chapter
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
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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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