Development of a surface plasmon resonance-based assay for characterizing PDZ domain interactions
AbstractPDZ domains are modular protein binding domains involved in many important processes near brain synapses, such as protein clustering, scaffolding and trafficking. The main interaction partner for PDZ domains is the C-terminal end of proteins, especially membrane proteins, such as G protein-coupled receptors (GPCRs) and ion channels. One of the best studied systems is the interaction between the first PDZ domain (PDZ1) of postsynaptic density protein 95 (PSD-95) and the C-terminal end of the NMDA receptor subunit 2B (NR2B). Both are highly concentrated in or very close to the membrane in the postsynaptic density.
We are developing a surface based platform for functional screening of PDZ domain interactions with membrane proteins. As a model system, we chose the model interaction between PSD-95 PDZ1 and a peptide mimicking the NR2B C-terminus. The surface plasmon resonance (SPR) studies are compared to data obtained with a bulk standard assay based on fluorescence polarization (FP). Two immobilization strategies were tested (random immobilization and SNAP-tag immobilization ), with similar results for Rmax and KD. The affinity of PDZ1 to the NR2B C-terminal end estimated from equilibrium SPR measurements was in good agreement with FP measurements and most of the literature. Using the same strategy, a novel interaction with the C-terminal end of the somatostatin receptor 1 (SSTR1) was characterized. Interestingly, SSTR1 showed affinity comparable to NR2B, but different binding kinetics, pointing out the potential of SPR-based screening platforms for functional assays.