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

More documents

Recommendations

Info

1780 F. Fernandes et al. / Biochimica et Biophysica Acta 1758 (2006) 1777–1786 Fig. 3. CD spectrum of peptide H4 incorporated in DOPC at an L/P of 50. Another method to study aggregation of peptides is to follow the quantum yield changes of the aromatic residues present. Fluorescence from tyrosine residues is known to be quenched by several mechanisms besides FRET, namely interactions with other residues side chains such as glutamate and aspartate, and uncharged forms of basic residues such as arginine, lysine and histidine, as well as electron transfer to peptide α-carbonyl groups [30,31]. Considering this, it can be expected that peptides forming aggregates will have a lower quantum yield than their monomeric counterparts. The quantum yield dependence of Tyr15 (corresponding to Tyr142 in the native protein [32]) from reconstituted peptides on the lipid composition is shown in Fig. 4. The presence of anionic phospholipids clearly increases the quantum yield of peptide H4. In liposome of pure zwitterionic phospholipids, the Tyr15 quantum yield trend is: DEuPC< DOPC
F. Fernandes et al. / Biochimica et Biophysica Acta 1758 (2006) 1777–1786 1781 Fig. 5. (A): Model used for FRET simulations. The donor (Tyr15) is located 6 Å from the center of the bilayer while the acceptors can be located at different planes, assuming a superficial location such as for NBD-DOPE (A 1 ) or more buried positions such as for the V-ATPase inhibitors (A 2 ). In case of aggregation the number of protein–protein contacts increases while protein–lipid contacts decrease, as a result, R e (exclusion distance) increases. (B) H4 fluorescence quenching of Tyr15 of peptide H4 due to FRET to NBD-DOPE in DOPC bilayers using a lipid to protein ratio of 50 (●) and 100 (○). Curve corresponds to an exclusion radius fit to the energy transfer data using equations 1–5 (—). A value of 9 Å was recovered for R e .(–––) FRET simulation for a random distribution of acceptors around a donor with an exclusion radius of 20 Å. n 2 is the superficial concentration of labeled phospholipids (molecules per Å 2 ). The range of NBD labeled phospholipid to total phospholipid ratios in this experiment was 0.4% to 2%. Inset: Overlap between tyrosine fluorescence emission ( ) and DOPE-NBD absorption spectrum (––), R 0 (Tyr-NBD) =22Å. On the basis of these results, the lipid composition chosen for the inhibitor-peptide binding studies by FRET measurements was 100% DOPC at an L/P of 100, as this lipid is able to favor the transmembrane orientation for peptide H4. The problem of lateral aggregation in the membrane nevertheless potentially remained, as in DOPC the tyrosine quantum yield was significantly lower than in liposomes containing anionic phospholipids. The possibility of aggregation could however also be assessed, as described below. A FRET experiment was performed in order to determine the size of the possible aggregates. Using the Tyr15 as a donor and NBD labeled DOPE as acceptors (NBD-DOPE), FRET efficiencies were obtained (Fig. 5B). Fitting the available theoretical models for energy transfer between donor and acceptors in different planes [34] to our experimental data, we can recover an averaged exclusion radius (R e ) of the donor, defined as the minimum distance between the Tyr15 and the NBD labeled phospholipids (Fig. 5A). For a monomeric peptide this value should be around 10 Å as this is approximately the sum of the radius of a α-helix backbone and that of a phospholipid molecule. Any significant increase from this value is likely to be reporting an extensive aggregation phenomenon. FRET efficiencies (E) are calculated from the degree of fluorescence emission quenching of the donor caused by the presence of acceptors. E ¼ 1 I DA I D ¼ 1 Z l 0 Z l i DA ðÞdt= t i 0 D ðÞdt t ð1Þ i DA ðtÞ ¼i D ðtÞ:q interplanar ðtÞ ð2Þ where I DA and I D are the steady-state fluorescence intensities of the donor in the presence and absence of acceptors respectively. i DA and i D are the donor decays in the presence and absence of acceptors. ρ interplanar is the FRET contribution arising from energy transfer to randomly distributed acceptors in two different planes from the donors (two monolayer leaflets) [34]. ( Z l p ) ( ffiffiffiffiffiffiffi 1 q interplanar ¼ exp 2n 2 pl1 2 l 1 2 1 expð tb 3 Z l þR2 e 1 a6 Þ p ) ffiffiffiffiffiffiffi 2 a 3 da exp 2n 2 pl2 2 l 2 2 1 expð tb 3 þR2 e 2 a6 Þ a 3 da 0 0 ð3Þ
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 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
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
Page 107 and 108: BINDING OF INHIBITORS TO A PUTATIVE
Page 109 and 110: BINDING OF INHIBITORS TO A PUTATIVE
Page 111: INTERACTION OF THE INDOLE CLASS OF
Page 114 and 115: 5272 Biochemistry, Vol. 45, No. 16,
Page 123: BINDING ASSAYS OF INHIBITORS TOWARD
Page 126 and 127: 1778 F. Fernandes et al. / Biochimi
Page 136 and 137: apparently the most significant as
Page 138 and 139: 110
Page 140 and 141: Introduction Quinolones are broad-s
Page 142 and 143: [9-12]. Having this into considerat
Page 144 and 145: any case, very similar, probably wi
Page 146 and 147: placement of the protein around the
Page 148 and 149: ⎛ II t ⎛ R ⎞ 0 ρ () t = exp
Page 150 and 151: APPENDIX - Derivation of the FRET r
Page 152 and 153: 2 2w J ( t) = '2 R − R + w / 1
Page 154 and 155: [14] L. Plançon, M. Chami, L. Lete
Page 156 and 157: acceptors) (Eqs. 4-6) (⋅-⋅-⋅)
Page 158 and 159: FIGURE 1A FIGURE 1B 130
Page 164 and 165: 136
Page 166 and 167: dynamin. Soon after, the same group
Page 168 and 169: 140
Page 170 and 171: 142
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
Page 178 and 179:
where η is the viscosity of the me
Page 180 and 181:
ound conformation of the BAR domain
Page 182 and 183:
19. Fernandes, F., L. M. S. Loura,
Page 184 and 185:
Figure Legends Fig.1: N-terminal am
Page 186 and 187:
FIG. 1A FIG.1B 17
Page 188 and 189:
FIG. 3A FIG. 3B 19
Page 190 and 191:
FIG.5A 21
Page 192 and 193:
FIG 6. 23
Page 194 and 195:
FIG. 8 A B 25
Page 196 and 197:
The signalling functions of PI(4,5)
Page 198 and 199:
172
Page 200 and 201:
174
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 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
show all

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

Create successful ePaper yourself

Delete template?

Save as template?