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

More documents

Recommendations

Info

FRET Study of Protein-Lipid Selectivity 349 FIGURE 4 Donor (DCIA-labeled protein) fluorescence quenching by energy transfer acceptor (18:1-(12:0-NBD)-PX), where X stands for the different headgroup structures, in pure bilayers of DOPC. (d), Experimental energy transfer efficiencies; (—), theoretical simulations obtained from the annular model for protein-lipid interaction using the fitted K S ; and (---), simulations for random distribution of acceptors (K S ¼ 1.0). (A) PC-labeled phospholipid (fitted K S ¼ 2.0); (B) PE-labeled phospholipid (fitted K S ¼ 2.0); (C) PG-labeled phospholipid (fitted K S ¼ 2.3); (D) PS-labeled phospholipid (fitted K S ¼ 2.7); and (E) PAlabeled phospholipid (fitted K S ¼ 3). ditions from a study of BODIPY labeled protein steady-state fluorescence emission (Fernandes et al., 2003). However, in that study much higher concentrations of protein were used when compared with the present one, and assuming the aggregation constants obtained in that work, only up to 5% at the very most of protein could be aggregated at the protein concentration used throughout this study. Therefore we can consider that our results report the phospholipid selectivity properties of the monomeric M13 major coat protein. The fitting of the annular model for protein-lipid interactions to the FRET data (Figs. 3 and 4), converged always to K S values above 1 (Table 1), as the energy transfer Biophysical Journal 87(1) 344–352
350 Fernandes et al. efficiencies (1 ÿ t DA =t D ) are above the expected value for random distribution of the labeled phospholipids. As our annular model assumes a random distribution outside the protein-lipid interface (which should be true for onecomponent bilayers in the liquid crystalline phase; Loura et al., 1996), this result is rationalized as an increase in local concentration of probe in the lipid annular shell around the protein. For (18:1) 2 -PE-NBD probe in DOPC ((18:1) 2 -PC) bilayers (Fig. 3 A), the value of K S was 1.4, pointing to almost complete randomization of the probe distribution in the bilayer, and therefore identical selectivity to the DOPC lipid. This was expected, because the probe acyl-chains are identical to the unlabeled lipid and allow a perfect hydrophobic matching of the protein. The results from Fig. 3, A–C, all report energy transfer efficiencies to the (18:1) 2 -PE-NBD probe, but in different bilayers of one lipid component. In DOPC bilayers the value of K S was 1.4 as discussed above, but in DMoPC ((14:1) 2 - PC) and DEuPC ((22:1) 2 -PC) bilayers the relative association constant values were 2.9 and 2.1, respectively, confirming a greater selectivity toward the hydrophobic matching unlabeled phospholipid (DOPC). M13 coat protein selectivity toward phospholipids with different headgroups Results from analysis of the data on M13 coat protein selectivity toward phospholipids headgroups are presented in Fig. 4. Clearly the anionic-labeled phospholipids exhibit larger selectivity to the lipid annular region around the protein, especially the 18:1-(12:0-NBD)-PA and 18:1-(12: 0-NBD)-PS probes (K S ¼ 3.0 and 2.7, respectively). The 18:1-(12:0-NBD)-PG probe presents an intermediate selectivity (K S ¼ 2.3), whereas 18:1-(12:0-NBD)-PC and 18: 1-(12:0-NBD)-PE have identical relative association constants (K S ¼ 2.0). The selectivity for anionic phospholipids must be a consequence of electrostatic interaction of these with the highly basic C-terminal domain of the protein, which contains four lysines. Overall the selectivity of annular lipid-M13 coat protein is not large, which is common for intrinsic membrane proteins (Lee, 2003). Additionally, it has been shown that selectivity of some proteins toward anionic lipids is significantly decreased in the presence of increasing ionic strength (Marsh and Horváth, 1998). In our case the ionic strength was kept high, because it is necessary to keep the protein in the monomeric state (Spruijt and Hemminga, 1991), and this further explains our results. Phospholipid selectivity ESR studies have already been performed with aggregated forms of M13 major coat protein (Peelen et al., 1992; Wolfs et al., 1989, Datema et al., 1987), resulting in similar selectivity patterns of M13 coat protein to phospholipid headgroups. Peelen et al. (1992), using a identical buffer type, ionic strength, and pH to that used in the present study, obtained the following relative association constants ratios (K S (PX)/K S (PC)) of M13 coat protein incorporated in 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC): K S (PA)/K S (PC) ¼ 1.6, K S (PS)/K S (PC) ¼ 1.2, K S (PG)/K S (PC) ¼ 1.1, and K S (PE)/K S (PC) ¼ 1. Overall the selectivity pattern is the same, and the relative association constants ratios are almost identical. The M13 coat protein was aggregated in that study, and according to the authors the number of first shell sites was five, that is, for each protein only a maximum of five lipids could be motionally restricted due to contact with the protein surface. For a monomeric helix, however, a value of 12 should be expected (Marsh and Horváth, 1998), and that was the number used in our model for the data analysis. Therefore it is particularly interesting that the ratios of the relative association constants remain almost identical. Apparently protein aggregation lowers the selectivity degree of each protein for phospholipids only through a decrease of available area for protein-lipid contacts but the relative association ratio with phospholipids of different headgroups remains the same. Even though the protein presents higher selectivity for the NBD-labeled phospholipids than for DOPC (K S (PC) ¼ 2.0) (possibly due to electrostatic interactions with the NBD at the bilayer interface), the result presented above clearly shows that the presence of NBD at the bilayer interface does not change significantly the relative association ratios of the phospholipids. Sanders et al. (1992) were not able to determine the selectivity of the M13 coat protein monomeric species toward phospholipids using ESR, because the monomer was not able to produce a sufficiently long-living boundary shell of lipids that could be detected by ESR spectroscopy. The fact that it was possible to clearly quantify relative association constants using FRET in the present study presents this technique as an alternative to ESR in protein-lipid studies. One important difference between the ESR and FRET techniques is that the latter is not restricted to the lipids adjacent to a given protein molecule. Not only labeled lipids in the first shell of lipids will be potential acceptors to a donor-labeled integral protein, but also the acceptors in the other lipid shells surrounding the protein will contribute to the final result. For that reason, this study also seems to confirm the hypothesis of selectivity to anionic phospholipids by the protein to largely confine itself to an annular shell of lipids in direct contact with the protein, in the case of the M13 major coat protein. In case that the annular region would extend beyond this first shell, our FRET analysis methodology (based in transfer to a single annular shell and also to the bulk) would recover substantially larger values for the relative association constants. Moreover, as commented above, our recovered K S /K S (PC) match those obtained from ESR measurements, which only detects immobilization of annular lipids upon incorporation of protein. The existence of a single annular Biophysical Journal 87(1) 344–352
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 160 and 161:
Page 162 and 163:
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 ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?