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.
Highlights
Attenuated total reflection Fourier transform infrared spectroscopy (ATR–FTIR) is one of the most powerful methods for recording infrared spectra of biological materials in general, and of biological membranes in particular
We demonstrate that sensors based on the BIA–ATR technology are capable of monitoring the binding of a single phosphate on such a large protein entity
Attenuated total reflection infrared (ATR–FTIR) spectra were obtained on a Bruker IFS 55 FTIR spectrophotometer (Ettlingen, Germany) equipped with a MCT detector at a resolution of 2 cm−1. 128 scans were accumulated for each spectrum
Summary
Attenuated total reflection Fourier transform infrared spectroscopy (ATR–FTIR) is one of the most powerful methods for recording infrared spectra of biological materials in general, and of biological membranes in particular. A. Goldsztein et al / Gastric ATPase phosphorylation/dephosphorylation to monitoring ligand binding, it provides a full spectrum characterizing both molecule nature and 3D structure. We recently demonstrated that chemical modification of germanium ATR crystals is possible and provides stable surfaces suitable for sensor applications [2,3,10].
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