Gastric ATPase phosphorylation/dephosphorylation monitored by new FTIR-based BIA-ATR biosensors

Biosensors are composite devices suitable for the investigation of receptor-ligand interactions. In this paper we present the specific application to a membrane embedded protein of a new sensor device, so-called BIA-ATR, based on Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy. It consists in a functionalised ATR germanium crystal whose surface has been covalently modified to adsorb a biomembrane. Detection of the ligand-receptor interaction is achieved using FTIR spectroscopy. We report the specific detection of the phosphorylation/dephosphorylation of the H+/K+ gastric ATPase. The H+, K+-ATPase is a particularly large protein entity. This glycosylated protein contains more than 1300 residues and is embedded in a lipid membrane. Yet we demonstrate that the BIA-ATR sensor is capable of monitoring the binding of a single phosphate on such a large protein entity. Furthermore, we also demonstrate the potential of the approach to monitor the kinetics of binding and dissociation of the ligand.