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

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drying into a film and ressuspension in an aqueous environment. Suspensions of liposomes prepared in this manner are composed by concentric bilayers or multilamellar vesicles (MLV’s). Due to this arrangement of the lipid vesicles, most of the lipid bilayer surface is buried inside the liposome, as only about 10 % is found in the outside surface (Yeagle, 1993). The application of MLV´s in photophysical studies can result in several difficulties. Due to the size of the MLV’s particles, scattering is very significant and for these reason, MLV´s are seldom used. There are different strategies for obtaining unilamellar liposomes. By applying ultrasonic power to suspensions of liposomes, unilamellar vesicles of small diameter (~30 Å), also called small unilamellar vesicle (SUV) are obtained. Due to the smaller size of the particles, scattering in suspensions of SUV’s is drastically reduced, however the curvature of SUV’s is much higher than the curvature of cell membranes, resulting in poor mimics of the properties of biomembranes. Larger unilamellar vesicles (LUV’s) can be produced either by dialysis of detergents, reverse phase evaporation, or fast extrusion through polycarbonate filters. The latter method is particularly useful due to the shorter times required when compared to the other ones, e.g., dialysis of detergents can take several days for efficient removal of detergent from liposomes (for a review, see Gennis, 1989). Much larger unilamellar vesicles (up to 300 µm) can also be prepared by gentle hydration and electroformation (Rodriguez et al., 2005). These vesicles are also known as giant unilamellar vesicles or GUV’s and are particularly suitable for microscopy applications as their size enables visualization and micromanipulation. Still, other types of membrane model systems exist. Lipid monolayers in water-air interface allow the study of the effect of the lateral surface pressure on membrane components and on the interactions between them. Black Lipid Membranes have also proven valuable in the study of electrical properties (Yeagle, 1993). 22
INTRODUCTION: LIPID-PROTEIN INTERACTIONS 2.LIPID-PROTEIN INTERACTIONS 2.1. Membrane protein reconstitution Even for the simplest organisms, membrane proteins in protein-lipid extracts are present in a very complex and heterogeneous environment, which can impair the possibility of conducting biophysical studies that require a simpler matrix for the protein (with absence of possible contaminants), and more controlled conditions. However, integral membrane proteins generally cannot be studied in homogeneous systems such as aqueous or organic solutions due to the complex solubility problems derived from their bitopic nature. In addition, the study of their properties in a isotropic medium, would not be biologically relevant. Systems are required that satisfy both the hydrophobicity of the membrane embedded sections of the protein as well as their hydrophilic domains. Liposomes are frequently used for such studies, since, as discussed in the previous section, they present good mimetic conditions of biomembranes. Liposomes containing reconstituted proteins are called proteoliposomes. In order to reconstitute membrane proteins in liposomes it is first necessary to purify and solubilize them. Neither of these procedures is trivial and detergents have proven invaluable tools in both. They meet the requirements of amphipatic structure necessary to solubilize the two different environments present in membrane proteins and these are frequently soluble in micellar structures. Detergents are classified according to the charge of the headgroup as ionic (cationic or anionic), non-ionic or zwitterionic. Examples of ionic detergents often used in membrane solubilization are sodium dodecyl sulphate (SDS) and bile acid salts. SDS is a linear chain detergent and a extremely powerful solubilizing agent for membrane proteins, however, it is also frequently denaturing. Protein renaturation is eventually possible under certain conditions after transfer to other medium (Dong et al., 1997). Bile acid salts are ionic detergents with steroidal groups for backbones. Due to the planar characteristics of the steroidal structure, instead of a proper headgroup they present a polar and an apolar face. These detergents are much weaker than SDS and more efficient in maintaining protein activity. Examples of bile acid salts are sodium cholate and sodium deoxycholate. Non-ionic detergents are also mild and relatively non-denaturating/non- 23
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 and 26: OUTLINE membranes with a distributi
Page 27: OUTLINE BAR domains (tubulation of
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 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
Page 77 and 78: PROTEIN-PROTEIN AND PROTEIN-LIPID I
Page 79: PROTEIN-PROTEIN AND PROTEIN-LIPID I
Page 83 and 84: 2430 Biophysical Journal Volume 85
Page 85 and 86: 2432 Fernandes et al. Coat protein
Page 87 and 88: 2434 Fernandes et al. leads to an a
Page 89 and 90: 2436 Fernandes et al. FIGURE 4 (A)
Page 91 and 92: 2438 Fernandes et al. section of th
Page 93 and 94: 2440 Fernandes et al. tein oligomer
Page 95: QUANTIFICATION OF PROTEIN-LIPID SEL
Page 98 and 99: FRET Study of Protein-Lipid Selecti
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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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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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