Investigations of phospholipid/cholesterol and cholesterol derivative interactions in model membranes
Author(s)
Trevenen, Alexandra H
Type
Thesis
Abstract
Multinuclear solid state MAS NMR (1H and 31P), 31P CSA solid state NMR, 2H
NMR and x-ray diffraction techniques have been used to compare the structure
and properties of DPPC: Andro and DPPC: 4β-hydroxycholesterol with that of
the properties known of DPPC: Chol mixtures in excess water. The formation
of the Lo phase is known to occur with PC: Chol mixtures with sufficient
concentrations of cholesterol. The formation and properties of the Lo phase was
looked at with the cholesterol derivatives Andro (lacking in the hydrocarbon tail
present in cholesterol) and 4β-hydroxycholesterol (possessing an extra hydroxyl
group adjacent to the one present at the headgroup region of cholesterol). The
Lo phase shows fluid-fluid immisibility when combined with the disordered Lα
phase. It is this heterogeneity that is believed to be important in the formation
of lipid rafts, which are thought to play a role in the function of living cells.
Cholesterol desorption from DOPC model membranes was examined using
methyl-β-cyclodextrin, which has a high affinity for cholesterol and facilitates
the mechanism of cholesterol desorption from lipid membranes. By running a
number of experiments, measuring cholesterol desorption from MLV’s and LUV’s a cholesterol flip-flop rate within DOPC model membranes was
measured using 1H solution state NMR.
A number of methods were also attempted to synthesise an asymmetric DOPC:
DPPC membranes. These included a split glass slides method and a modified
version of an emulsion method as described by Pautot et al. lipid asymmetry
with respect to the split glass slides and emulsion methods was to be observed
by NMR, where any cholesterol asymmetry formed via desorption by methyl-β-
cyclodextrin was to be evaluated via the use of paramagnetic NMR
experiments, however no asymmetry was observed in either experiment.
NMR and x-ray diffraction techniques have been used to compare the structure
and properties of DPPC: Andro and DPPC: 4β-hydroxycholesterol with that of
the properties known of DPPC: Chol mixtures in excess water. The formation
of the Lo phase is known to occur with PC: Chol mixtures with sufficient
concentrations of cholesterol. The formation and properties of the Lo phase was
looked at with the cholesterol derivatives Andro (lacking in the hydrocarbon tail
present in cholesterol) and 4β-hydroxycholesterol (possessing an extra hydroxyl
group adjacent to the one present at the headgroup region of cholesterol). The
Lo phase shows fluid-fluid immisibility when combined with the disordered Lα
phase. It is this heterogeneity that is believed to be important in the formation
of lipid rafts, which are thought to play a role in the function of living cells.
Cholesterol desorption from DOPC model membranes was examined using
methyl-β-cyclodextrin, which has a high affinity for cholesterol and facilitates
the mechanism of cholesterol desorption from lipid membranes. By running a
number of experiments, measuring cholesterol desorption from MLV’s and LUV’s a cholesterol flip-flop rate within DOPC model membranes was
measured using 1H solution state NMR.
A number of methods were also attempted to synthesise an asymmetric DOPC:
DPPC membranes. These included a split glass slides method and a modified
version of an emulsion method as described by Pautot et al. lipid asymmetry
with respect to the split glass slides and emulsion methods was to be observed
by NMR, where any cholesterol asymmetry formed via desorption by methyl-β-
cyclodextrin was to be evaluated via the use of paramagnetic NMR
experiments, however no asymmetry was observed in either experiment.
Date Issued
2009-09
Date Awarded
2010-03
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Magee, Tony
Seddon, John
Sponsor
EPSRC
Creator
Trevenen, Alexandra H
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)