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

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Steady-state fluorescence measurements were carried out with an SLM-Aminco 8100 Series 2 spectrofluorimeter described in detail elsewhere (13). Fluorescence anisotropies were determined as described in (14). Time-resolved fluorescence measurements of H0-BAR-EDANS were carried out with a time-correlated single-photon timing system, which is also described elsewhere (13). For steady-state fluorescence anisotropies and time resolved fluorescence measurements with H0-NBAR- EDANS, the excitation and emission wavelengths were 340 and 460 nm, respectively. For time-resolved fluorescence measurements with H0-NBAR-FITC, the excitation and emission wavelengths were 470 and 525 nm, respectively. Analysis of fluorescence intensity and anisotropy decays were carried out as previously described (15,16). All measurements were performed at room temperature. Transmission electron microscopy (TEM) TEM was carried out using a Jeol (Tokyo, Japan) Electron Microscope 100SX, operated at 60kV. Samples were placed over copper grids covered with carbon membrane. Negative staining was performed with uranyl acetate 1% in water. Total lipid concentration of the mixtures was varied (ranging from 0.05 mM to 0.5 mM). Results The N-terminal segment of the N-BAR domain is 100% alpha-helical in anionic liposomes. CD measurements were performed on the unlabeled H0-NBAR peptide while in the absence of liposomes, in the presence of zwitterionic liposomes (POPC) and anionic liposomes (POPG) (Fig.2). CD measurements of the labeled H0-NBAR peptides (both H0-NBAR- EDANS and H0-NBAR-FITC) produced the same results, confirming that labeling did not change the structure of H0-NBAR (results not shown). Deconvolution of the obtained spectrum with the CDNN CD Spectra Deconvolution v. 2.1 software revealed a predominantly unstructured peptide in the absence of lipids and in the presence of 1mM of POPC. After addition of 1mM of POPG the peptide presented more than 80 % alpha-helical structure, compatible with the expected amphipathic nature of the N-terminal segment of the N-BAR domain while interacting with lipid membranes. Interaction of H0-NBAR with lipid bilayers is only dependent on lipid charge. The fluorescence emission spectrum from H0-NBAR-EDANS is practically unaffected upon interaction with lipid bilayers, as the wavelength of maximum emission intensity remains the same and only a small broadening in the lower wavelength range of the spectrum is visible (results not shown). EDANS emission spectrum is extremely sensitive to the environment (17) and this result is an indication that the EDANS fluorophore is located in the hydrophilic face of the amphipathic helix and that it remains fully exposed to the aqueous environment. There was however a subtle difference in quantum yields (20% higher in the presence of anionic phospholipids) and a significant change in fluorescence anisotropy, from = 0.022 in buffer to > 0.06 in the presence of anionic phospholipids. This change in fluorescence anisotropy 5
is the result of the immobilization of the peptide in the lipid bilayer and can be used to quantify the partition of H0-NBAR to bilayers from equation 1 (18), where W is the fluorescence anisotropy in water, L is the fluorescence anisotropy in the bilayer, φ W is the fluorescence quantum yield in water, φ L is the fluorescence quantum yield in the membrane, γ L is the molar volume of the lipid, [L] is the lipid concentration and K p is the lipid/water partition coefficient. Figure 3A shows the dependence of on the lipid concentration for different phospholipids. It is clear that partition to POPC liposomes is extremely low when compared to the partition observed for both anionic phospholipids POPS and POPG. The results of fitting Equation 1 to the data in Figure 3 are presented in Table I. Partition of H0-NBAR to POPS and POPG liposomes is identical suggesting that the nature of the anionic phospholipid is irrelevant for H0-NBAR interaction with liposomes. Further evidence for the absence of a specific interaction with a class of lipids, can be obtained from a FRET assay, where lipids (e.g. PS, PA, and PG) are derivatized with an acceptor (NBD) for Förster resonance energy transfer (FRET) from EDANS, and dispersed (2% mol/mol concentration) in a POPG matrix. FRET efficiencies (E) are obtained from the extent of fluorescence emission quenching of the donor induced by the presence of acceptors, [ ] [ L] φw⋅ < r > + K ⋅γ ⋅ L ⋅φ ⋅< r > < r >= 1+ K ⋅γ ⋅ w P L L L P L (1) IDA E = 1− = 1− I D ∞ ∫ DA 0 ∞ 0 i ∫ i D () t () t (2) where I DA and I D are the the steady-state fluorescence intensities of the donor in the presence and absence of acceptors respectively, and i DA (t) and i D (t) are the donor fluorescence decays in the presence and absence of acceptors respectively. In case there was a specific interaction between H0-NBAR-EDANS and the NBDlabeled phospholipid, the peptide would be incorporated in the vicinity of that lipid and FRET would increase. Since there is also liposome fusion induced by H0-NBAR-EDANS (see later), the available two dimensional FRET formalisms may not be applicable for retrieving quantitative information about H0-NBAR-EDANS selectivity for a specific phospholipid (19), and a more qualitative approach was used. The results are shown in the inset of Fig. 3A. It is clear that no significant difference exists between E obtained with any of the anionic phospholipids. The small differences in FRET efficiencies are a result of the lack of selectivity of H0-NBAR-EDANS for specific phospholipids. Partition of H0-NBAR-EDANS to lipid membranes is also insensitive to the dimensions of the liposomes and hence insensitive to the degree of curvature of the bilayer in this range of liposome size (Fig. 3B). Even though the values of K p recovered for larger liposomes are slightly higher (as seen in Table I), the differences are within the errors of the fits and thus non significant. Residual partition to POPC liposomes was detected. After long incubation times (overnight), an increase of fusion of POPC liposomes loaded with H0-NBAR was evident relative to blank POPC vesicles. This was clear from a FRET experiment carried out with a mixture of liposomes loaded with 2% NBD-DOPE (donor) and liposomes loaded with 2% 6
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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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Page 123: BINDING ASSAYS OF INHIBITORS TOWARD
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Page 136 and 137: apparently the most significant as
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Page 140 and 141: Introduction Quinolones are broad-s
Page 142 and 143: [9-12]. Having this into considerat
Page 144 and 145: any case, very similar, probably wi
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 176 and 177: Rho-DOPE (acceptor). The increase o
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 200 and 201: 174
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
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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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