Gastric ATPase phosphorylation/dephosphorylation monitored by new FTIR-based BIA–ATR biosensors
Issue title: From Molecule to Tissue: XIII European Conference on the Spectroscopy of Biological Molecules, Palermo, Italy, August 28–September 2, 2009, Part 2 of 2
Affiliations: Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles, Belgium | Laboratoire de Physique des Surfaces et des Interfaces, Université de Mons, Mons, Belgium | Physique des Matériaux et Optique, Université de Mons, Mons, Belgium | Unité de Chimie Organique et Médicinale, Université Catholique de Louvain, Bâtiment Lavoisier, Louvain-la-Neuve, Belgium
Note: [] Corresponding author: Andréa Goldsztein, Center for Structural Biology and Bioinformatics, Laboratory for the Structure and Function of Biological Membranes, Campus Plaine CP206/02, Université Libre de Bruxelles, Bld du Triomphe 2, CP206/2, B1050 Brussels, Belgium. Tel.: +32 2 650 53 62; Fax: +32 2 650 53 82; E-mail: angoldsz@ulb.ac.be.
Abstract: 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.
