Biophysical studies of membrane proteins/peptides. Interaction with ...
Biophysical studies of membrane proteins/peptides. Interaction with ...
Biophysical studies of membrane proteins/peptides. Interaction with ...
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CONCLUSIONS<br />
Chapter VI – Clustering <strong>of</strong> PI(4,5)P2 in Fluid PC Bilayers.<br />
Domain enrichment <strong>of</strong> PI(4,5)P 2 is expected to occur in vivo. Nevertheless the<br />
process must require strong binding to agents (<strong>proteins</strong>) that present a strong affinity for<br />
incorporation in cholesterol enriched domains. Clustering <strong>of</strong> charged phospholipids in<br />
the gel state is certainly possible as energetic requirements resulting from packing<br />
restrictions in this state can surpass the energetic penalty resulting from electrostatic<br />
repulsion between the clustered lipids. However, the same type <strong>of</strong> phenomena is<br />
expected to be highly unlikely in fluid bilayers, where packing restrictions are not so<br />
severe.<br />
The results from this study allowed us to conclude that PI(4,5)P 2 is homogeneously<br />
distributed in fluid PC bilayers. FRET <strong>studies</strong> ruled out the existence <strong>of</strong> any large scale<br />
domain (dimensions larger than 40 Å) enriched in PI(4,5)P 2 , while energy migration<br />
and fluorescence self-quenching <strong>studies</strong> <strong>of</strong> a fluorescently labelled PI(4,5)P 2 clearly<br />
indicate absence <strong>of</strong> strong short-range clustering <strong>of</strong> the lipid.<br />
Another important result from this study are the differences in water/bilayer<br />
partition efficiencies obtained for different protonation states <strong>of</strong> PI(4,5)P 2 . One possible<br />
explanation for this observation is that the micellar structure <strong>of</strong> PI(4,5)P 2 is destabilized<br />
at the completely deprotonated state (at low lamellar lipid concentrations, a micellar<br />
population <strong>of</strong> PI(4,5)P 2 is expected to be in equilibrium <strong>with</strong> a bilayer inserted<br />
population). These differences in partition behaviour can result in erroneous<br />
rationalization <strong>of</strong> fluorescent data, specially taking into account that the most popular<br />
fluorescently labelled PI(4,5)P 2 in the market presents very short acyl-chains, and will<br />
present even smaller partition efficiencies that the ones determined here for a long chain<br />
fluorescently labelled PI(4,5)P 2 .<br />
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