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

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FINAL CONSIDERATIONS AND PROSPECTS VIII FINAL CONSIDERATIONS AND PROSPECTS Biophysical studies are likely the only possibility of direct structural and dynamic characterization of the type of interactions addressed in this work. The recurrent difficulties in membrane protein expression and purification were responsible for the popularity of the application of highly sensitive fluorescence techniques in studies of membrane proteins. The presence of aromatic amino acids in proteins allows the application of fluorescence techniques without requirement for labelling with a extrinsic fluorophore. Still, when necessary, the developments in synthesis of fluorophores and labelling techniques, provide the researcher with great flexibility in the process of choosing the best conditions for fluorescence methodologies. Overall, the studies presented here are a demonstration of the potential of biophysical studies, particularly fluorescence spectroscopy studies, in the characterization of interactions between components of biomembranes. In this work, the use of membrane model systems, allowed for the simplification of the number of variables in the experiments. This is essential in studies that focus on the characterization of very specific interactions. Apart from the technical difficulties inherent to in vivo studies (huge heterogeneity of samples, background fluorescence, light scattering, low fluorescence intensity, etc.), the presence of a large number of uncontrolled (and sometimes unknown) variables is highly likely to hinder the possibility of data rationalization. The information recovered from studies with less complex model systems can then be used for a more clear rationalization of data gathered from in vivo studies. The new FRET methodologies developed here, notably the methodology for quantification of protein-lipid selectivity, are expected to be of great use in the future as they are readily applicable to different systems. The FRET methodologies for quantification of drug-protein binding will require only some straightforward adaptations to the geometric constraints of the systems to be studied. 189
Some of the biological questions debated here are still open. Recently, a model for the binding site of V-ATPase inhibitors in the c-subunit was proposed (Bowman et al., 2006), confirming that residues in at least three different transmembrane domains were required for effective bafilomycin inhibition. However the picture was not completely clear, as residues in opposite sides of the same helix were found to be relevant for bafilomycin binding pointing to indirect effects of some residues in the interaction with the inhibitor. Although, clinical trials with SB242784 were not started due to possible adverse effects (Nikura et al., 2005), a derivative of this molecule was shown recently to potentiate the effect of an cytotoxic/anti-tumor drug likely due to inhibition of V- ATPases in tumor cells (Petrangoline et al., 2006). The study of the mechanisms behind clustering of PI(4,5)P 2 into cholesterol dependent domains in biomembranes is a very promising field. It is clear that PI(4,5)P 2 should not spontaneously cluster or partitionate to liquid ordered domains, and this behaviour must be connected to specific protein-lipid interactions. In this way, the study of the lipid membrane partition properties of proteins known to bind PI(4,5)P 2 might give us some insight into these mechanisms. The relevance of acylation characteristics (number and length of acyl-chains in the protein), for membrane protein organization in biomembranes, is a problem of great biological relevance. The use of fluorescence imaging techniques in the study of protein-lipid interactions can add additional layers of information to the results of non-space resolution biophysical studies. The biophysical studies presented here relied mostly on macroscopic observables, and although this type of data is, as demonstrated, highly enriched in information, some of it is lost by averaging of fluorophore populations, while imaging techniques allow for resolution of different populations of fluorophores. Therefore, the application of the methodologies derived and described here to fluorescence imaging data could help identify features of interactions between membrane components which go undetected in the macroscopic data. In the limit, single-molecule techniques could also be used. Imaging techniques are obviously very useful in the observation of large scale changes (µm scale) in the lateral distribution of membrane components and are a useful complement to FRET methodologies, which are able to probe deviations to homogeneity in the nanometer scale. Additionally, FRET studies under the microscope are also carried out, allowing the direct comparison between macroscopic and microscopic data. 190
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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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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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Page 172 and 173: Introduction Control of membrane re
Page 174 and 175: Steady-state fluorescence measureme
Page 176 and 177: Rho-DOPE (acceptor). The increase o
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Page 184 and 185: Figure Legends Fig.1: N-terminal am
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Page 196 and 197: The signalling functions of PI(4,5)
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Page 202 and 203: zoxadiazol (NBD)-PC were obtained f
Page 204 and 205: Fig. 3. Fluorescence decays of diph
Page 206 and 207: CONCLUSIONS VII CONCLUSIONS Chapter
Page 208 and 209: CONCLUSIONS constants recovered by
Page 210 and 211: CONCLUSIONS Chapter IV ‐ Binding
Page 212 and 213: CONCLUSIONS Chapter VI - Clustering
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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