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

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Figure I.9 – Depiction of the several possibilities for association of proteins with biomembranes. Protein domains in the exoplasmic side of the membrane are frequently glycosilated and can interact with the extracellular matrix. Protein domains on the cytoplasmic side on the other hand are responsible for the coupling of the cytoskeleton network with the lipid membrane (From Lodish et al., 2000). Membrane proteins on the extracellular side of the plasma membrane generally bind other molecules, including external signalling proteins, ions, small metabolites, and adhesion molecules in other cells. Protein domains within the plasma membrane, mainly those that are part of channels and pores, are generally responsible for transport of molecules across the bilayer, while peripheral protein membranes in the cytosolic side of the plasma membrane are responsible for many different functions (Lodish et al., 2000). The possibilities of secondary structure available for membrane spanning protein domains are very limited. Only two structures are available, the alpha-helix and the beta-barrel (Figure I.10). These structures have a common characteristic. In both, the peptide bonds of the aminoacids are hydrogen bonded. In fact, if this were not the case, insertion in the highly apolar environment would be energetically unfavourable, even for the most hydrophobic amino acids. The energetic cost of partitioning a peptide bond into a highly apolar phase, is significantly larger than the free energy reduction associated with insertion of the Trp side chain in the same environment (White and Wimley, 1999). Therefore, only a polypeptide segment with complete backbonebackbone hydrogen bonding can insert in the highly hydrophobic core of the bilayer, and the alpha-helix and the beta-barrel are the two structures that satisfy this condition. 18
INTRODUCTION: BIOMEMBRANES A B Figure I.10 – Two secondary structures available for membrane spanning protein domains, the alphahelix (A) and the beta-barrel (B). The amino acids in an alpha-helix are arranged in a right-handed helical structure. The dimensions of the alpha helix are 5,4 Å wide (if accounting for aminoacid side chains the alpha helix is about 10 Å wide). Each aminoacid correspond to a 100º turn, and consequently for each turn, 3.6 aminoacids are required. Each aminoacid also corresponds to a translation across the helical axis of 1,5 Å, and each amine group of the peptide bound forms a hydrogen bond with the carbonyl group of the pepide bound four aminoacids earlier. Beta barrels are large beta sheet structures for which the last strand is hydrogen bonded with the first, creating in this way a closed structure. These structures are commonly found in porins (see chapter IV). The beta-sheet structure is based on the side-by-side hydrogen bonded arrangement of stretchs of amino acids linearly extended. Hydrogen bonding between peptide bonds is essential for formation of TM protein segments, however it is not sufficient. The sum of the free energy reduction from insertion of the side chains of the polypeptide must surpass the energetic cost of burying the hydrogen bonded peptide bonds. This is only possible in the presence of highly hydrophobic aminoacids, and in the absence of to many hydrophilic ones, particularly charged residues. Octanol is frequently regarded as a suitable model for studying the insertion of molecules in a hydrophobic medium such as the hydrocarbon core of the bilayer. Figure I.11 reproduces the results obtained for the free energy of transfer of whole aminoacids (including the peptide bond), from water to the octanol phase, and to the interface region (polar environment) of POPC bilayers. 19
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
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Page 75 and 76: PROTEIN-PROTEIN AND PROTEIN-LIPID I
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Page 83 and 84: 2430 Biophysical Journal Volume 85
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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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