Site specific immobilization of the peptidoglycan synthase PBP1B on a surface plasmon resonance chip surface

Abstract

Surface Plasmon Resonance (SPR) is one of the most powerful label free methods to determine kinetic parameters of molecular interactions in real time and in a high sensitive way. Penicillin binding proteins (PBPs) are peptidoglycan synthesis enzymes present in most bacteria. The established protocols to analyze interactions of PBPs by SPR involves immobilization to an ampicillin coated chip surface, a β-lactam antibiotic mimicking its substrate, forming a covalent complex with the PBPs transpeptidase (TP) active site. In this way, interactions are measured of PBPs with a substrate bound TP domain, potentially affecting interactions near the TPase active site. Furthermore, in vivo PBPs are anchored in the inner membrane by an N-terminal transmembrane helix, and hence, immobilization via their C-terminal TPase domain gives an orientation contrary to the in vivo situation. We designed a new procedure, immobilizing a PBP using copper-free click chemistry, via an azide incorporated in its N-terminus. We present a proof-of-principle study, in which we use this new method to immobilize Escherichia coli PBP1B on an SPR chip surface and show its use for the analysis of the well characterized interaction of PBP1B with LpoB. The site specific incorporation of the azide creates the possibility to determine the protein orientation, resulting in a homogeneous immobilization on the chip surface. This method can be used to study the topology-dependent interactions of any (membrane) protein.