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Title: Locating binding sites of the general anaesthetic propofol using photoaffinity labelling
Author: Yip, Grace Mei Shan
ISNI:       0000 0004 2735 6082
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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3-(5’-isopropyl-6’-hydroxyphenyl)-3-trifluoromethyl-diazirine (photofol), a new drug developed in our laboratory, is structurally homologus to the general anaesthetic propofol with one of its isopropyl groups being substituted for a trifluoromethyl-diazirine group. Photofol was shown to act in a similar manner to propofol, albeit with a three-fold reduction in potency on inducing a loss of righting reflex in rats and with reduced modulation of GABA-evoked currents on α1 β2 γ2s GABAA receptors for the concentration tested. The soluble protein human serum albumin (HSA) and the pentameric ligand-gated ion channel GLIC from the prokaryote Gloeobacter violaceus were used to test the utility of photofol as a photoaffinity label. Both proteins have been crystallised with propofol to produce high-resolution X-ray structures (Bhattacharya et al., 2000; Nury et al., 2011). Two samples of the photolabel – one containing naturally occurring hydrogen atoms and the other a bespoke proportion of different isotopes with between zero and two deuterium atoms – were used and this novel approach was successfully demonstrated to aid the analysis of mass spectra through their differences in isotopic distribution. Photolabelling HSA and GLIC followed by enzyme cleavage, liquid chromatography-mass spectrometry and tandem mass spectrometry identified the binding of photofol to six different amino acids. Photofol labelled all three protofol-binding sites of HSA identified by X-ray crystallography. Peptides that are part of the propofol-binding site in the GLIC transmembrane domain reduced in ionisation intensity upon photolabelling, suggesting that photofol had bound to this vicinity. Furthermore, photolabelling of binding sites at the interfaces between subunits in the extracellular domain was reproducibly observed, particularly at lysine-183. Electrophysiology-couple mutagenesis studies are currently underway to confirm to propofol binding to the site, where binding to another general anaesthetic, ketamine, has recently been shown by high-resolution structures (Pan et al., 2012a). In conclusion, the work presented herein indicates that photofol is a suitable photolabel for locating for locating binding sites of propofol.
Supervisor: Franks, Nick Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral