Prof. Dr. Joachim Jose

ChemBIon project description

ChemBIon project descriptions
Autodisplay of the NMDAR GluN1/2A ligand binding domains
More recently, the ligand binding domains (LBDs) of GluN1 and of GluN2A were expressed in soluble form in E. coli and were shown to form functional dimers, again proven by kinetic studies. Co-crystallization experiments unveiled the binding mode of negative allosteric modulators (NAMs) e.g. TCN-201, which address a pocket, formed at the interface of both subunits. Conformational changes after binding the agonists glutamate and glycine with or without NAMs provided a clear concept for the observed allosteric inhibition of NMDAR. Objective: The goal of this project is a) the co-expression of the LBDs of GluN1 and GluN2A on the bacterial cell surface and b) to show the functionality by kinetic studies with known compounds. This will enable c) to establish a new screening method for antagonists and negative allosteric modulators of NMDAR by a fluorescence derivative of TCN-201.
MST based assay for HCN channel activator identification
Microscale thermophoresis (MST) is a new method for the rapid measuring of KD values of two binding partners in µL solutions.3 The motion of molecules within a temperature gradient is called Ludwig-Soret-effect or thermophoresis. MST determines the different motion of a molecule with or without bound ligand within a moderate temperature gradient between 23 -25°C, which is induced by an IR laser beam. For this purpose, different concentrations of the ligand are added to a fixed concentration of the fluorescence labelled binding partner, and for each concentration the thermophoresis is determined. The resulting curve, thermophoresis in dependence of ligand concentration, yields the binding constant. Using this strategy, KD values can be determined with minimal amounts of binding partners within minutes. More recently, we could show that human target proteins can be labelled by click chemistry and used in soluble or surface displayed form for the determination of protein-small molecule interactions and protein-protein interactions
Objective: The aim of this project is the development of a rapid screening assay for HCN4 channel activators using microscale thermophoresis (MST) based assay.