Revealing the Interaction of Binding Protein with ABC Transporter by Fluorescence Correlation Spectroscopy

Abstract

ATP binding cassette (ABC) transporters can be found in all organism form bacteria to man. Dysfunction of these transporters is often associated with diseases, like multi drug-resistance in cancer cells. These transporters translocate substances across the cellular plasma membrane under the expense of ATP, consist of dimers of ATPase subunits known as the ATP binding cassette and of two transmembrane domains (TMDs).The histidine permease (HisQMP2) of S. typhimirium is a well characterised classic canonical ABC importer. For substrate translocation HisJ (binding protein (BP) with specificity for histidine) or LAO (BP with specificity for lysine/arginine/ornithine) are required. The BP interacts with the extracellular loops of the TMD and the binding constants for both proteins were found to be in a micro molar range. To assess the conformational dynamics of BP-TMD complexation during the catalytic cycle of substrate translocation, a Fluorescense Correlation Spectroscopy based binding assay is designed to investigate the dependency of this interaction on the catalytic state of the transporter. HisQMP2 is functionally reconstituted into large unilamellar vesicles and a monocysteine variant of the BP is chemically labelled with Alexa Fluor 488. By trapping the transporter in different steps of ATP hydrolysis, the change in diffusion rates of the fluorescent BP is supposed to reveal the dynamics of the BP association during substrate translocation.