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

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zoxadiazol (NBD)-PC were obtained from Avanti Polar Lipids (Birmingham, AL). NBD-PI(4,5)P 2 was from Echelon Biosciences (Salt Lake City, UT). Diphenylhexatriene (DPH) was obtained from Molecular Probes (Leiden, The Netherlands). Other fine chemicals were from Merck (Darmstadt, Germany). Liposome reconstitution procedure The desired amounts of phospholipids were mixed in chloroform-methanol (1:2) and dried under an N 2(g) flow. The sample was then kept in a vacuum overnight. Liposomes were prepared with 20 mM HEPES, 100 mM NaCl buffer at pH 8.4 and 7.1 and with 20 mM sodium citrate, 100 mM NaCl buffer at pH 4.8. The hydration step was performed with the addition of buffer followed by freeze-thaw cycles. Anisotropy measurements were performed with large unilamellar vesicles produced by extrusion through polycarbonate filters with a pore size of 100 nm (14). to quantify the effective fraction of NBD-PI(4,5)P 2 incorporated in liposomes. NBD quantum yield decreases with hydration, and clustering of NBD-PI(4,5)P 2 leads to NBD selfquenching (20). Consequently, it was not surprising that NBD-PI(4,5)P 2 in buffer (micellar state) exhibited quantum yields z100 times smaller than when incorporated in PC liposomes (lamellar state) (Fig. 1). This difference allowed us to use fluorescence intensity as a tool to estimate the extent of water/bilayer partition for NBD-PI(4,5)P 2 . From equation 3 (21), the extent of partition can be quantified into partition coefficients (K p ) I 5 I w 1 K P 3 g L 3 [L] 3 I L 1 1 K P 3 g L 3 [L] (Eq: 3) Fluorescence measurements Steady-state fluorescence measurements were carried out with an SLM-Aminco 8100 Series 2 spectrofluorimeter described in detail elsewhere (15). Steady-state anisotropies were determined according to Lakowicz (16). NBD-PC and NBD-PI(4,5)P 2 anisotropies were recorded with excitation and emission wavelengths of 460 and 540 nm, respectively, with spectral bandwidths of 4 nm. All measurements were performed at room temperature. Fluorescence decay measurements of DPH (donor) were carried out with a time-correlated single-photon timing system, which is described elsewhere (15). All measurements were performed at room temperature. Excitation and emission wavelengths were 340 and 430 nm, respectively. In the FRET experiments, the donor decays in the presence of acceptors [i DA (t)] can be described as i DA (t) 5 i D (t) 3r interplanar (t) (Eq: 1) where i DA (t) and i D (t) are the fluorescence decays of the donor in the presence and absence of acceptors, respectively. r interplanar is the FRET contribution arising from energy transfer to randomly distributed acceptors in two different planes from the donors (two monolayer leaflets) (17) 8 l 9 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi l 2 1 R >< 2 e r interplanar 5 exp 24 n 2 3p3l 2 1 2 exp(2t 3 b # 3 3a 6 ) >= a 3 da >: 0 >; Downloaded from www.jlr.org by on September 3, 2007 (Eq: 2) where b 5 (R 0 2 /l) 2 H D 21/3 ,R 0 is the Förster radius, n 2 is the acceptor density in each leaflet, and l is the distance between the plane of the donor and the two planes of acceptors. The Förster radius was calculated as described elsewhere (18). RESULTS NBD-PI(4,5)P 2 partition to lipid bilayers When using a labeled phospholipid as the acceptor in a FRET study, the energy transfer efficiency will depend strongly on its concentration inside these vesicles. As PI(4,5)P 2 is known to generally form micelles in the aqueous medium (19), it is essential in the type of measurements mentioned above to determine whetherallNBD-PI(4,5)P 2 incorporates in the liposomes and, if that is not the case, Fig. 1. Fluorescence intensity (I F ) of phosphatidylinositol-(4,5)- bisphosphate [PI(4,5)P 2 ] as a function of POPC (A) and 1-palmitoyl- 2-oleoylphosphatidylglycerol (POPG) (B) concentration. Closed circles, pH 8.4; open circles, pH 7.1; closed squares, pH 4.8. The curves are fits of equation 3 to the experimental data, allowing the determination of water/bilayer partition coefficients (K p ). 7-Nitro- 2-1,3-benzoxadiazol (NBD)-PI(4,5)P 2 concentration 5 1 mM. a.u., arbitrary units. 1522 Journal of Lipid Research Volume 47, 2006
where I is the fluorescence intensity, I W and I l are the fluorescence intensities in buffer and in liposomes, respectively, g l is the lipid molar volume, and [l] is the lipid concentration. The curves obtained when the NBD-PI(4,5)P 2 fluorescence intensities were plotted versus the lipid concentrations are shown in Fig. 1. Three different pH values (8.4, 7.1, and 4.8) corresponding to three different protonation states of PI(4,5)P 2 (for the micellar state Pk a2 5 6.7 for the 49 position and 7.6 for the 59 position) were studied. The results for the different pH values were clearly different when using POPC bilayers: a larger extent of partition was observed when pH 8.4 was used (corresponding to a total charge of 25), whereas partition for pH 7.1 (24) was almost identical to that for pH 4.8 (23). For POPG, the partition was much less dependent on pH. The K p values obtained at pH 8.4, 7.1, and 4.8 for water/ POPC partition were (5.38 6 0.60) 3 10 4 , (1.84 6 0.14) 3 10 4 , and (2.18 6 0.18) 3 10 4 , respectively, whereas for micelle/POPG partition, these were (1.84 6 0.33) 3 10 4 , (2.29 6 0.38) 3 10 4 , and (1.47 6 0.18) 3 10 3 , respectively. Clustering of NBD-PI(4,5)P 2 in POPC Clustering of NBD-PI(4,5)P 2 inside the POPC matrix would result in a decrease of fluorescence intensity attributable to self-quenching of NBD and in a decrease in fluorescence anisotropy attributable to energy migration (energy homotransfer) inside the clusters (20). The two parameters were studied for increasing NBD-PI(4,5)P 2 concentrations inside a POPC matrix, as shown in Fig. 2. NBD-PI(4,5)P 2 concentrations were corrected for the partition coefficients determined above. As a control, the fluorescence intensity and anisotropies of NBD-PC in POPC and of NBD-PI(4,5)P 2 in DPPC were compared with the data for NBD-PI(4,5)P 2 in POPC. NBD-PC in POPC is homogeneously distributed, whereas in DPPC, clustering is expected for NBD-PI(4,5)P 2 as a result of gel-fluid phase separation. It is clear that NBD-PI(4,5)P 2 and NBD-PC have identical clustering behavior in POPC. The degree of selfquenching and energy migration of both lipids is almost identical and much smaller than for NBD-PI(4,5)P 2 in DPPC at room temperature, at which clustering is observed as a result of packing restraints in the DPPC gel matrix (22). We also performed a FRET experiment, using for this purpose the fluorescence decay of the donor. The reason for a time-dependent approach instead of a steady-state approach is that the kinetics of the donor decay gives information on both the distribution (homogeneity/heterogeneity) and concentration of acceptors (15), whereas the steady-state approach is unable to distinguish between the two. Again, we chose POPC as the matrix lipid, the donor was DPH, which is known to have no preference between lipid phases (23), and the acceptor was NBD- PI(4,5)P 2 . In this system, in the case of NBD-PI(4,5)P 2 clustering, two populations of DPH would be detectable, one residing in a NBD-PI(4,5)P 2 -rich site and the other in a NBD-PI(4,5)P 2 -poor area of the bilayer. We globally fitted the FRET model for a single discrete concentration of acceptors (homogeneous distribution of acceptors) (equations 2, 3) to the data in Fig. 3, and for all pH values used (only results for pH 8.4 are shown), good quality fits were obtained (global Chi-square , 1.5). The acceptor concentrations were the only free parameter during these fits, and the recovered values matched closely (610%) the bilayer concentrations expected from the partition coefficients determined above, again confirming the absence of NBD-PI(4,5)P 2 clustering. DISCUSSION Compartmentalization of PI(4,5)P 2 has been reported several times, but its origin had always been attributed to Downloaded from www.jlr.org by on September 3, 2007 Fig. 2. Fluorescence intensity (I F ) of NBD-PI(4,5)P 2 in POPC (closed circles), POPG (open circles), and 1,2-dipalmitoylphosphatidycholine (DPPC; closed diamonds) bilayers and of phosphatidylcholine NBD-PC in POPC (closed squares). Inset: Fluorescence anisotropy (,r.) of NBD-PI(4,5)P 2 in POPC (closed circles) and DPPC (open diamonds) bilayers and of NBD-PC in POPC (closed squares). Error bars represent SD. All measurements were performed at pH 8.4. Total lipid concentration 5 0.2 mM. a.u., absorbance units. Absence of PI(4,5)P 2 clustering in fluid PC 1523
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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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1778 F. Fernandes et al. / Biochimi
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apparently the most significant as
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110
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Introduction Quinolones are broad-s
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[9-12]. Having this into considerat
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any case, very similar, probably wi
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placement of the protein around the
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⎛ II t ⎛ R ⎞ 0 ρ () t = exp
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APPENDIX - Derivation of the FRET r
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Page 156 and 157: acceptors) (Eqs. 4-6) (⋅-⋅-⋅)
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Page 172 and 173: Introduction Control of membrane re
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Page 206 and 207: CONCLUSIONS VII CONCLUSIONS Chapter
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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
Page 226 and 227: BIBLIOGRAPHY Pluschke, G., Hirota,
Page 228 and 229: BIBLIOGRAPHY Sperotto, M. M., and M
Page 230: BIBLIOGRAPHY Williamson, I. M., Alv
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