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Title: The use of enzymes, isomerisable lipid analogues and small molecules to modulate the structure and dynamics of lipid bilayer
Author: Batchelor, Rebecca
ISNI:       0000 0004 2705 9096
Awarding Body: University of Hull
Current Institution: University of Hull
Date of Award: 2010
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A series of experiments were carried out to assess the effects and interactions of isomerisable lipid analogues, small molecules, and enzyme catalysed substrates on the structure and dynamics of lipid bilayers. Modulation of Enzyme Activity Using an Azobenzene-Containing Lipid Analogue – An azobenzene-containing phosphocholine lipid analogue (bis-Azo-PC) was synthesised and incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes containing diacylglycerol kinase (DGK) in order to assess the effect of photoisomerisation of the azobenzene group on the activity of the protein. The increase in volume of bis-Azo-PC in the cis state should have a significant effect on protein activity as bilayer tension increases. Protein activity was measured using an enzyme activity assay. The difference in specific activity between the trans and cis states of the azobenzene was too small to discern a significant effect on protein activity. Lipid Phase Behaviour – The phase behaviour of 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine-d62 (d62-DPPC) samples containing cholesterol and the serotonin receptor agonist drugs quipazine and LY-165,135 was analysed using proton and deuterium NMR spectroscopy and differential scanning calorimetry (DSC). The effect of quipazine and LY-165,135 on d62-DPPC was an elongation of the gel-to-fluid lamellar phase transition, lowering of the start of the phase transition, and disappearance of the ripple phase observed in d62-DPPC alone. The effect of cholesterol on d62-DPPC was a smoothing of the phase transition due to the presence of the liquid-ordered phase. Spectra of d62-DPPC, cholesterol and quipazine also show the presence of the liquid-ordered phase. Spectra of d62-DPPC and cholesterol with LY-165,135 show that the drug widens the temperature range over which the liquid-ordered phase exists. These results indicate that the presence of small molecules such as serotonin-receptor agonists can have a significant effect on the phase behaviour of the lipid bilayer. Analysis of Drug Localisation and Orientation in a Model Membrane by NMR Spectroscopic Methods – NOESY cross-relaxation rates were used to generate location probabilities for serotonin-receptor agonist drugs in d62-DPPC bilayers with and without cholesterol. Both drugs were found to locate in the interface region of the bilayer, although they had difference orientations relative to the bilayer normal; quipazine was oriented parallel to the d62-DPPC molecules, while LY-165,135 was oriented perpendicular to the lipid. The presence of cholesterol was found to affect the drugs‟ positions in the bilayer. Quipazine was pushed further towards the centre of the bilayer (the acyl chain termini) when the sample was in the fluid lamellar phase. The effect on LY-165,135, conversely, was to push it to towards the headgroup region as the fluid lamellar phase approached. The drug interactions with cholesterol during the mixed [lo + ld] phases indicate that these compounds may be partitioning into the liquid-ordered phase under these conditions. Use of an Enzyme Substrate to Investigate Bilayer Dynamics – DROSS NMR, 31P NMR and fluorescence emission spectroscopy were used to analyse the effect of catalysing an enzyme substrate on a lipid bilayer. 1,2-Dioctanoyl-sn-glycerol (DOG) is a substrate of DGK. DGK phosphorylates DGK in the presence of adenosine triphosphate (ATP), producing phosphatidic acid (PA). DROSS was used to generate order parameters while fluorescence emission spectroscopy was used to give an indication of bilayer tension. Bilayer tension and order parameters were lower for the sample containing PA co-dissolved with DOPC relative to the sample containing catalysed PA. This is due to sequestration of the catalysed PA in the immediate vicinity of the protein rather than an even distribution throughout the bilayer (as observed for co-dissolved PA), which affects the physical properties of the bilayer.
Supervisor: Lorch, Mark ; Archibald, Steve Sponsor: Not available
Qualification Name: Not available Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Chemistry