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

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FRET Study of Protein-Lipid Selectivity 347 lateral separation between the probes was assumed to be 8 Å. The estimated d was then 20.5 and 21.4 Å for NBD derivatized in the headgroup and acyl-chain, respectively. For the DMoPC and DEuPC bilayers the values used for l were 15.4 and 22.4 Å, respectively, as for each additional carbon in the phospholipid chain in the liquid crystalline phase the bilayer thickness increases 1.75 Å (Lewis and Engelman, 1983). Considering a hexagonal-type geometry for the proteinlipid arrangement (Fig. 1 b), each protein will be surrounded by 12 annular lipids. In bilayers composed by both labeled and unlabeled phospholipids, these 12 sites will be available for both of them. The probability (m) of one of these sites to be occupied by labeled phospholipid is given by m ¼ K S n NBD n NBD 1 n lipid : (9) Here, n NBD is the concentration of labeled lipid, and n lipid is the concentration of unlabeled lipid. K S is the relative association constant, which reports the relative affinity of the labeled and unlabeled phospholipid. Using a binomial distribution we can calculate the probability of each occupation number (0–12 sites occupied simultaneously by labeled lipid), and finally the FRET contribution arising from energy transfer to annular lipids, n¼12 r annular ðtÞ ¼ + e ÿnk Tt 12 m n ð1 ÿ mÞ 12ÿn : (10) n¼0 n The FRET contribution from energy transfer to acceptors randomly distributed outside the annular region in two different planes at the same distance to the donor plane (from the center of the bilayer to both leaflets) is given by Davenport et al. (1985) as RESULTS M13 coat protein selectivity toward phospholipids with different hydrophobic thickness The DCIA-labeled protein quantum yield was determined (f ¼ 0.41). Using Eqs. 5 and 6, and assuming k 2 ¼ 2/3 (the isotropic dynamic limit) and n ¼ 1.4 (Davenport et al., 1985), R 0 ¼ 39.3 Å is obtained for the DCIA-NBD FRET pair (Fig. 2). The value k 2 ¼ 2/3 was used, because for fluorophores in the center of a liquid crystalline bilayer, the rotational freedom should be sufficiently high to randomize orientations (for a detailed discussion see Loura et al., 1996). FRET selectivity studies were performed in bilayers of one lipid component (DOPC, DMoPC, or DEuPC) using T36C M13 major coat protein mutant labeled with DCIA as the donor and (18:1) 2 -PE-NBD (1,2-dioleoyl-sn-glycero- 3-phosphoethanolamine derivatized with NBD at the headgroup) as the acceptor. The donor fluorescence intensities ratio (t DA =t D ), which is related to the energy transfer efficiency, decreases upon increasing the acceptor (Eq. 3). The results are presented in Fig. 3. The results of fitting the derived formalism to the data are also shown in this figure, and the corresponding K S values are summarized in Table 1. M13 coat protein selectivity toward phospholipids with different headgroups Energy transfer studies were also performed to determine the selectivity properties of M13 coat protein toward phospholipids with different headgroups. Again, the donor was T36C coat protein mutant labeled with coumarin (DCIA), but various probes were used as acceptors, all studies being made in DOPC vesicles. The probes used as acceptors were r random ( Z pffiffiffiffiffiffiffiffi ) l ¼ exp ÿ4n 2 pl 2 l 2 1 R 2 1 ÿ expðÿtb 3 a 6 Þ e a 3 da ; 0 (11) where b ¼ðR 2 0 =lÞ2 t ÿ1=3 D ; n 2 is the acceptor density in each leaflet, l is the distance between the plane of the donors and the planes of acceptors, and R e is the distance between the protein axis and the second lipid shell (exclusion distance for bulk-located acceptors). In the present system, l is the unlabeled lipid bilayer thickness, and the exclusion distance is 16 Å assuming a radii of 5 Å and 4.5 Å for the protein and the phospholipid, respectively; see Fig. 1 b). The value n 2 must be corrected for the presence of labeled lipid in the annular region, which therefore is not part of the randomly distributed acceptors pool. FIGURE 2 Corrected emission spectrum of DCIA-labeled M13 major coat protein (—), and corrected excitation spectrum of NBD-derivatized phospholipid (---). Biophysical Journal 87(1) 344–352
348 Fernandes et al. FIGURE 3 Donor (DCIA-labeled protein) fluorescence quenching by energy transfer acceptor ((18:1) 2 -PE-NBD) in pure phosphatidylcholine bilayers with different hydrophobic thickness. (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) Labeled protein incorporated in DOPC (fitted K S ¼ 1.4); (B) labeled protein incorporated in DMoPC (fitted K S ¼ 2.9); and (C) labeled protein incorporated in DEuPC (fitted K S ¼ 2.1). phospholipids of identical acyl-chains (18:1 and 12:0) and different headgroups (PC, PE, PS, PG, and PA) classes, derivatized with NBD at the 12:0 chain. The results are shown in Fig. 4, together with the model fits. Table 1 summarizes the recovered K S values. M13 coat protein could be submitted to irreversible aggregation. This hypothesis has been ruled out for both lipids in recent studies (Meijer et al., 2001; Fernandes et al., 2003). Nevertheless, M13 major coat protein was shown recently by us to aggregate reversibly while in these con- DISCUSSION M13 coat protein selectivity toward phospholipids with different hydrophobic thickness The M13 coat protein is known to form large irreversible aggregates under specific conditions. These aggregates are not found in vivo, and therefore are regarded as an artifact (Hemminga et al., 1993). Due to their b-sheet conformation, they are detected by CD spectroscopy, and because of the hydrophobic mismatch between the protein and DEuPC (longer) or DMoPC (shorter) lipids, it was possible that while incorporated in pure bilayers of these components, the TABLE 1 Labeled phospholipids’ relative association constants toward M13 major coat protein Labeled phospholipid Bilayer composition K S K S /K S (PC)* ((18:1) 2 -PE-NBD) DOPC (18:1) 2 PC 1.4 — ((18:1) 2 -PE-NBD) DEuPC (22:1) 2 PC 2.1 — ((18:1) 2 -PE-NBD) DMoPC (14:1) 2 PC 2.9 — (18:1-(12:0-NBD)-PE) DOPC 2.0 1.0 (18:1-(12:0-NBD)-PC) DOPC 2.0 1.0 (18:1-(12:0-NBD)-PG) DOPC 2.3 1.1 (18:1-(12:0-NBD)-PS) DOPC 2.7 1.3 (18:1-(12:0-NBD)-PA) DOPC 3.0 1.5 *K S (PC) is the relative association constant of (18:1-(12:0-NBD)-PC). 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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Page 83 and 84: 2430 Biophysical Journal Volume 85
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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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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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