A Fluorescence Based Method for Studying the Membrane Topology of the Anti-Apoptotic Protein BCL-XL

Abstract

Bcl-XL is a membrane-associated protein that inhibits programmed cell death (apoptosis) in mammalian cells. Very little is known about the membrane topology of Bel-XL or how its association with membranes contributes to its function. It was the aim of this thesis to use fluorescence spectroscopy to investigate the location of a specific amino acid ofBcl-XL relative to the membrane. Bel-XL purified from E. coli could bind both to large unilamellar vesicles and endoplasmic reticulum (ER) microsomes isolated from canine pancreas. A cysteine residue at position 151 in Bcl-XL could be covalently labelled with the environmentally sensitive fluorescent molecule NBD. Emission intensity measurements in the presence and absence of membranes, combined with aqueous and lipophilic quenching experiments, indicate that Cys 151 is inserted into the interior of the membrane bilayer when Bcl-XL is bound to membranes. The methods outlined in this thesis form the basis for an experimental system that can be used to determine the membrane topology ofBclXL under a variety of conditions.