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

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1782 F. Fernandes et al. / Biochimica et Biophysica Acta 1758 (2006) 1777–1786 where b i =(R 0 2 /l i ) 2 τ D −1/3 , R 0 is the Förster radius, n 2 is the acceptor density in each leaflet, and l 1 and l 2 are the distance between the plane of the donors and the two planes of acceptors. Using Eqs. (1)–(3), theoretical expectations for FRET efficiency in a random distribution of acceptors can be calculated and converted to I DA /I D ratios. The Förster radius is given by: R 0 ¼ 0:2108ðJj 2 n 4 / D Þ 1=6 ð4Þ where J is the spectral overlap integral, κ 2 is the orientation factor, n is the refractive index of the medium, and ϕ D is the donor quantum yield. J is calculated as: Z J ¼ f ðkÞeðkÞk 4 dk ð5Þ where f(λ) is the normalized emission spectra of the donor and ε(λ) is the absorption spectra of the acceptor. The numeric factor in Eq. (4) assumes nm units for the wavelength λ and Å units for R 0 . Assuming the already determined value for Tyr15 position in the bilayer (6 Å from the center) and the distance from the center of the bilayer of the NBD moiety in NBD labeled phospholipids (19 Å) [35], l 1 and l 2 were determined to be 13 and 25 Å, respectively. Using Eqs. (4) and (5) the Förster radius of the Tyr-NBD donor–acceptor pair was determined to be R 0 =22 Å. In the fitting procedure, these values are kept fixed and the only parameter being fitted is R e. The results from the experiment along with the curve fitted from the theoretical model, are presented in Fig. 5B. Both sets of data (L/P=50 and 100) were fitted with a value of R e =9 Å, matching the expectation for a monomeric peptide. In case there were aggregates at the protein concentrations used these must be small enough so that the distances between Tyr15 and the surrounding phospholipids are not affected. From Eqs. (1)–(3) and using the same parameter values described above as well as R e =20 Å (large aggregates), a FRET simulation was performed for the same system and the obtained donor quenching curve is compared to our experimental values in Fig. 5B. 4.2. Peptide inhibitor binding studies Both bafilomycin A 1 and SB 242784 absorb in the UV region and can act as acceptors for tyrosine in energy transfer experiments (spectra are shown in Fig. 6 and 7). The Förster radius for the Tyr-bafilomycin A 1 donor–acceptor pair is 20 Å, whereas for the Tyr- SB 242784 pair it is 24 Å (Eq. (4)). A 3 mM concentration of lipid was used to ensure an almost complete incorporation of inhibitors in the bilayer. SB 242784 partition coefficient to DOPC bilayers is 1.20×10 4 [36], and at the lipid concentration used 97% of the inhibitor molecules are incorporated in the bilayer. The fraction of inhibitors not incorporated in the vesicles was taken into account on the acceptor concentrations plot in Fig. 7, which depicts the Tyr15 quenching via energy transfer to SB 242784. In contrast to SB 242784, bafilomycin A 1 is not a fluorescent molecule and partition coefficients could not be determined using photophysical techniques. Overestimation of its partition coefficient could lead to an underestimation of bafilomycin-peptide binding constants. However, it was recently showed by EPR of spin-labeled lipids that the macrolide molecule concanamycin A, another powerful V-ATPase inhibitor with very similar structure to bafilomycin A 1 , readily incorporates in lipid membranes [37]. Thus, it is to be expected from this result and from the high hydrophobic character of the molecule that the incorporation of bafilomycin A 1 at the lipid concentrations used is close to 100%. On the inhibitor-peptide binding assays, the L/P was kept at 100 in order to ensure minimum levels of aggregation. Experimental FRET efficiencies obtained with both peptide H4/SB 242784 and peptide H4/bafilomycin A 1 donor/acceptor pairs (Eq. (1)), are compared to theoretical expectations for a random distribution of acceptors (the scenario in which there is an absence of binding) obtained from Eqs. (1) and (2) (see Figs. 6 and 7). The position of SB 242784 in DOPC bilayers was already determined from selective quenching methodology (parallax method) to be 12.8 Å from the center of the bilayer [36], but the position of the bafilomycin A 1 chromophore was impossible to determine by the same methodology since this molecule is not fluorescent. In Fig. 6A, the simulations corresponding to different positions of the bafilomycin A 1 chromophore inside the bilayer are shown, together with the experimental data. It is clear that even when assuming that the Tyr15 is located on the same bilayer plane as the bafilomycin A 1 chromophore (6 Å from the center of the bilayer), in a situation of maximal energy transfer efficiency, the experimental energy transfer efficiencies cannot be solely explained by the unbound population of inhibitor molecules (i.e. random distribution of acceptors) and a fraction of peptide H4 must be binding bafilomycin A 1 . It is difficult to precisely quantify the binding constant (K b ) for this process due to the uncertainty relative to the bafilomycin A 1 chromophore position in the bilayer, but a lower and a higher limit can be determined assuming the closest and furthest position possible for bafilomycin A 1 and Tyr15 (corresponding respectively to a position in the center and in the surface of the bilayer for bafilomycin A 1 ), and also that peptide H4/bafilomycin A 1 complexes are completely non-fluorescent (Eqs. (6) and (7)). This last assumption is valid, since for a contact interaction either by Förster type or other transfer (exchange) mechanism, the efficiency of transfer would be 100%. The equations valid for the FRET efficiency in a scenario of peptide H4-bafilomycin A 1 1:1 complex formation are given below: E EXP ¼ E random þ H4 Baf ð1 E random Þ ð6Þ ½H4Š T
F. Fernandes et al. / Biochimica et Biophysica Acta 1758 (2006) 1777–1786 1783 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ½H4 Baf Š ¼ 1 K bð½Baf Š T þ½H4Š T Þþ ð1 þ K b ð½Baf Š T þ½H4Š T ÞÞ 2 4ðKb 2½Baf Š T ½H4Š T Þ ð7Þ ½H4Š T ð 2K b Þ½H4Š T where [H4-Baf] is the effective concentration (molecules per lipid volume as the binding is confined to the lipid phase) of the peptide–bafilomycin A 1 complex, and, [H4] T and [Baf] T are respectively, the effective analytical concentrations of the peptide and of bafilomycin A 1 . E EXP is the energy transfer efficiencies obtained experimentally and E random is the theoretical energy transfer efficiency assuming a random distribution of acceptors (Eqs. (1)–(3)). In case binding is detected, the use of effective bafilomycin A 1 concentrations will result in lower binding constants as compared to the ones of bulk concentrations, but the binding constants obtained in this way have a larger physical meaning as they will not change with lipid concentration. Anyway they can be easily interconverted. Assuming a position for bafilomycin A 1 in the center of the bilayer, the fitting procedure (Fig. 6A) recovered K b =10.5±1.32 M − 1 (mol per volume of lipid). When the bafilomycin A 1 position was fixed to 20 Å from the center of the bilayer (i.e. at the surface), the value recovered was K b =36.2±2.8 M − 1 . Therefore, the binding constant upper and lower bounds for the peptide H4–bafilomycin A 1 complex are 10.5±1.32 13.72±3.05 M − 1 . Fig. 6. (A) Fluorescence emission quenching of Tyr15 of peptide H4 by FRET to bafilomycin A 1 (●). Theoretical expectations (Eqs. (1–3)) for FRET in the absence of inhibitorbindingtopeptideH4assumingpositionsforbafilomycinA 1 of12.5Å(⋯),8.5Å(–––),and4.5Å(—) fromthecenterofthebilayer.( )Fittingofequations6 and 7 to FRET data assuming a bafilomycin A 1 position of 4.5 Å from the center of the bilayer. The interval 10.5±1.34
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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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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
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Page 89 and 90: 2436 Fernandes et al. FIGURE 4 (A)
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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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Page 140 and 141: Introduction Quinolones are broad-s
Page 142 and 143: [9-12]. Having this into considerat
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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) (⋅-⋅-⋅)
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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
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Page 176 and 177: Rho-DOPE (acceptor). The increase o
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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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