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

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was used in binding studies for the V-ATPase inhibitors bafilomycin and SB242784. This transmembrane domain is expected to comprise the binding site for the studied inhibitors. SB242784 was developed to be used as a potential drug for treatment of osteoporosis. This inhibitor presents selectivity for the osteoclastic form of the enzyme, while bafilomycin is extremely toxic due to its lack of selectivity. FRET was again used (a tyrosine residue of the peptide was used as a donor and the inhibitors as acceptors) to determine binding, and the results indicate a weak binding of the chosen peptide with bafilomycin whereas binding of the peptide to SB242784 was not detected. Overall, the results indicate that the V-ATPase inhibitor binding site is likely not formed only by the 4 th transmembrane segment of the c-subunit of V-ATPase, but is the result of contributions from other transmembrane domains, reflecting a more complex view of the inhibitory mechanism of V-ATPase than originally proposed. In Chapter IV, a binding study was conducted between ciprofloxacin (CP), a quinolone antibiotic, and a purified trimer of the outer membrane porin, OmpF. CP requires interactions with OmpF for efficient entry into the cell. In this case, the native protein structure was used instead of the minimalist approach described in Chapter III. FRET was again applied, this time making use of the presence of two types of tryptophans of OmpF (as donors) and the UV absorbing properties of CP (acceptor). Fluorescence from intrinsic amino acids of large proteins in FRET can be difficult to analyze, since each donor (fluorescent amino acid) population is expected to sense a different population of acceptors. Donors closer to the protein periphery, can e.g., be in closer proximity to acceptors than donors in the core of the protein. With this in mind, a FRET methodology suitable for application to FRET studies with multi-donor proteins was developed according to the geometric restrictions of the OmpF system. This model allowed the recovery of a range for the binding constants of the OmpF-CP association process. Due to the large dimensions of the OmpF trimer, depending on the site of inhibitor binding assumed in the model for FRET data analysis, the retrieved binding efficiency can be different. Two limiting binding sites were considered, and comparing the results from our analysis with the results obtained with an independent method, it was possible to conclude that the binding site for CP in OmpF is likely to be displaced from the center of the trimer and closer to the periphery. In Chapter V, the interaction of the N-terminal amphipatic peptide of a N-BAR domain with model membranes was studied. Several authors hypothesized that this segment of N-BAR domain contributed to the membrane remodelling properties of N-
OUTLINE BAR domains (tubulation of spherical liposomes) by inserting deeply into the bilayer core, driving monolayer asymmetry and thereby forcing a change in the normal curvature of the bilayer. We tested this hypothesis by following the interaction of the corresponding peptide with membrane model systems. Again, fluorescence spectroscopy methodologies were used and complemented with the application of electron microscopy to investigate changes in liposome morphology upon binding of the peptide. It is concluded that binding to liposomes is essentially electrostatic, ruling out the frequently hypothesized role of this protein segment to operate through the stabilization of the membrane bound conformation of BAR domains via hydrophobic short-range interactions with the bilayer core. Nevertheless, the results of a FRET study clearly indicate the tendency of the peptide to oligomerize in the lipid membrane environment, supporting a recently proposed function of this segment as a mediator of aggregation between BAR domain dimers. Electron microscopy measurements were not able to detect significant morphology changes in the liposomes upon addition of peptides. Finally, clustering behaviour of phosphatidylinositol-4,5-bisphophate (PI(4,5)P 2 ) in phosphatidylcholine bilayers was studied (Chapter VI) in order to test the hypothesis of spontaneous aggregation of this lipid in a PC matrix. Both fluorescence self-quenching data obtained with a fluorescently labelled PI(4,5)P 2 , and a FRET study using this lipid as a donor to a homogeneously distributed membrane probe, unequivocally confirmed a homogeneous distribution of PI(4,5)P 2 in the lipid bilayer. In the same study, it is demonstrated that different protonation states of PI(4,5)P 2 correspond to different water/lipid partition coefficients, a phenomenon that can be of significant biological relevance. xxv
Page 1: UNIVERSIDADE TÉCNICA DE LISBOA INS
Page 4 and 5: Fernandes, F., Loura, L. M. S., Fed
Page 6 and 7: Ao Pedro e Hugo, amigos de longa da
Page 8 and 9: 2.2. - Peptides as models 2.3. - Am
Page 11 and 12: ABBREVIATIONS AND SYMBOL LIST ABBRE
Page 13: RESUMO RESUMO As biomembranas são
Page 17 and 18: SINOPSE SINOPSE Nas últimas duas d
Page 19 and 20: SINOPSE podem fornecer informação
Page 21 and 22: SINOPSE aceitantes. Dadores mais pr
Page 23 and 24: OUTLINE OUTLINE The last two decade
Page 25: OUTLINE membranes with a distributi
Page 30 and 31: ion diffusion, as the energy requir
Page 32 and 33: acid. If no more groups are linked
Page 34 and 35: sphingomyelin, the most abundant sp
Page 36 and 37: signal for neighbouring cells to ph
Page 38 and 39: functional role in process such as
Page 40 and 41: in the L α phase, while lateral di
Page 42 and 43: 1.7. Lateral heterogeneity in lipid
Page 44 and 45: the vesicle through bilayer deforma
Page 46 and 47: Figure I.9 - Depiction of the sever
Page 48 and 49: Figure I.11 - Experimentally obtain
Page 50 and 51: drying into a film and ressuspensio
Page 52 and 53: deactivating agents. Zwitterionic d
Page 54 and 55: amphipatic helices (see Section 2.3
Page 56 and 57: size corresponding to the hydrophob
Page 58 and 59: changes abruptly in the interfacial
Page 60 and 61: thickness of the bilayer (Section 1
Page 62 and 63: some α-helical membrane proteins a
Page 64 and 65: The formation of a lipid population
Page 66 and 67: homogeneous distribution of lipids
Page 68 and 69: Figure I.20 - Relative binding cons
Page 70 and 71: 2.9. Lipid phase preferential parti
Page 72 and 73: In Figure I.21, theoretical simulat
Page 75 and 76: PROTEIN-PROTEIN AND PROTEIN-LIPID I
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PROTEIN-PROTEIN AND PROTEIN-LIPID I
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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 118 and 119:
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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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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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