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

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FRET Study of Protein-Lipid Selectivity 349 FIGURE 4 Donor (DCIA-labeled protein) fluorescence quenching by energy transfer acceptor (18:1-(12:0-NBD)-PX), where X stands for the different headgroup structures, in pure bilayers of DOPC. (d), Experimental energy transfer efficiencies; (—), theoretical simulations obtained from the annular model for protein-lipid interaction using the fitted K S ; and (---), simulations for random distribution of acceptors (K S ¼ 1.0). (A) PC-labeled phospholipid (fitted K S ¼ 2.0); (B) PE-labeled phospholipid (fitted K S ¼ 2.0); (C) PG-labeled phospholipid (fitted K S ¼ 2.3); (D) PS-labeled phospholipid (fitted K S ¼ 2.7); and (E) PAlabeled phospholipid (fitted K S ¼ 3). ditions from a study of BODIPY labeled protein steady-state fluorescence emission (Fernandes et al., 2003). However, in that study much higher concentrations of protein were used when compared with the present one, and assuming the aggregation constants obtained in that work, only up to 5% at the very most of protein could be aggregated at the protein concentration used throughout this study. Therefore we can consider that our results report the phospholipid selectivity properties of the monomeric M13 major coat protein. The fitting of the annular model for protein-lipid interactions to the FRET data (Figs. 3 and 4), converged always to K S values above 1 (Table 1), as the energy transfer Biophysical Journal 87(1) 344–352
350 Fernandes et al. efficiencies (1 ÿ t DA =t D ) are above the expected value for random distribution of the labeled phospholipids. As our annular model assumes a random distribution outside the protein-lipid interface (which should be true for onecomponent bilayers in the liquid crystalline phase; Loura et al., 1996), this result is rationalized as an increase in local concentration of probe in the lipid annular shell around the protein. For (18:1) 2 -PE-NBD probe in DOPC ((18:1) 2 -PC) bilayers (Fig. 3 A), the value of K S was 1.4, pointing to almost complete randomization of the probe distribution in the bilayer, and therefore identical selectivity to the DOPC lipid. This was expected, because the probe acyl-chains are identical to the unlabeled lipid and allow a perfect hydrophobic matching of the protein. The results from Fig. 3, A–C, all report energy transfer efficiencies to the (18:1) 2 -PE-NBD probe, but in different bilayers of one lipid component. In DOPC bilayers the value of K S was 1.4 as discussed above, but in DMoPC ((14:1) 2 - PC) and DEuPC ((22:1) 2 -PC) bilayers the relative association constant values were 2.9 and 2.1, respectively, confirming a greater selectivity toward the hydrophobic matching unlabeled phospholipid (DOPC). M13 coat protein selectivity toward phospholipids with different headgroups Results from analysis of the data on M13 coat protein selectivity toward phospholipids headgroups are presented in Fig. 4. Clearly the anionic-labeled phospholipids exhibit larger selectivity to the lipid annular region around the protein, especially the 18:1-(12:0-NBD)-PA and 18:1-(12: 0-NBD)-PS probes (K S ¼ 3.0 and 2.7, respectively). The 18:1-(12:0-NBD)-PG probe presents an intermediate selectivity (K S ¼ 2.3), whereas 18:1-(12:0-NBD)-PC and 18: 1-(12:0-NBD)-PE have identical relative association constants (K S ¼ 2.0). The selectivity for anionic phospholipids must be a consequence of electrostatic interaction of these with the highly basic C-terminal domain of the protein, which contains four lysines. Overall the selectivity of annular lipid-M13 coat protein is not large, which is common for intrinsic membrane proteins (Lee, 2003). Additionally, it has been shown that selectivity of some proteins toward anionic lipids is significantly decreased in the presence of increasing ionic strength (Marsh and Horváth, 1998). In our case the ionic strength was kept high, because it is necessary to keep the protein in the monomeric state (Spruijt and Hemminga, 1991), and this further explains our results. Phospholipid selectivity ESR studies have already been performed with aggregated forms of M13 major coat protein (Peelen et al., 1992; Wolfs et al., 1989, Datema et al., 1987), resulting in similar selectivity patterns of M13 coat protein to phospholipid headgroups. Peelen et al. (1992), using a identical buffer type, ionic strength, and pH to that used in the present study, obtained the following relative association constants ratios (K S (PX)/K S (PC)) of M13 coat protein incorporated in 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC): K S (PA)/K S (PC) ¼ 1.6, K S (PS)/K S (PC) ¼ 1.2, K S (PG)/K S (PC) ¼ 1.1, and K S (PE)/K S (PC) ¼ 1. Overall the selectivity pattern is the same, and the relative association constants ratios are almost identical. The M13 coat protein was aggregated in that study, and according to the authors the number of first shell sites was five, that is, for each protein only a maximum of five lipids could be motionally restricted due to contact with the protein surface. For a monomeric helix, however, a value of 12 should be expected (Marsh and Horváth, 1998), and that was the number used in our model for the data analysis. Therefore it is particularly interesting that the ratios of the relative association constants remain almost identical. Apparently protein aggregation lowers the selectivity degree of each protein for phospholipids only through a decrease of available area for protein-lipid contacts but the relative association ratio with phospholipids of different headgroups remains the same. Even though the protein presents higher selectivity for the NBD-labeled phospholipids than for DOPC (K S (PC) ¼ 2.0) (possibly due to electrostatic interactions with the NBD at the bilayer interface), the result presented above clearly shows that the presence of NBD at the bilayer interface does not change significantly the relative association ratios of the phospholipids. Sanders et al. (1992) were not able to determine the selectivity of the M13 coat protein monomeric species toward phospholipids using ESR, because the monomer was not able to produce a sufficiently long-living boundary shell of lipids that could be detected by ESR spectroscopy. The fact that it was possible to clearly quantify relative association constants using FRET in the present study presents this technique as an alternative to ESR in protein-lipid studies. One important difference between the ESR and FRET techniques is that the latter is not restricted to the lipids adjacent to a given protein molecule. Not only labeled lipids in the first shell of lipids will be potential acceptors to a donor-labeled integral protein, but also the acceptors in the other lipid shells surrounding the protein will contribute to the final result. For that reason, this study also seems to confirm the hypothesis of selectivity to anionic phospholipids by the protein to largely confine itself to an annular shell of lipids in direct contact with the protein, in the case of the M13 major coat protein. In case that the annular region would extend beyond this first shell, our FRET analysis methodology (based in transfer to a single annular shell and also to the bulk) would recover substantially larger values for the relative association constants. Moreover, as commented above, our recovered K S /K S (PC) match those obtained from ESR measurements, which only detects immobilization of annular lipids upon incorporation of protein. The existence of a single annular Biophysical Journal 87(1) 344–352
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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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Page 75 and 76: 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 95: QUANTIFICATION OF PROTEIN-LIPID SEL
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Page 123: BINDING ASSAYS OF INHIBITORS TOWARD
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Page 140 and 141: Introduction Quinolones are broad-s
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Page 150 and 151: 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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