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 the kinetic parameters of molecular interactions in real time and in a highly sensitive way. Penicillin‐binding proteins (PBPs) are peptidoglycan synthesis enzymes present in most bacteria. Established protocols to analyze interactions of PBPs by SPR involve immobilization to an ampicillin‐coated chip surface (a β‐lactam antibiotic mimicking its substrate), thereby forming a covalent complex with the PBPs transpeptidase (TP) active site. However, PBP interactions measured with a substrate‐bound TP domain potentially affect 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 at the C‐terminal TPase domain gives an orientation contrary to the in vivo situation. We designed a new procedure: immobilization of PBP by copper‐free click chemistry at an azide incorporated in the N terminus. In a proof‐of‐principle study, we immobilized Escherichia coli PBP1B on an SPR chip surface and used this for the analysis of the well‐characterized interaction of PBP1B with LpoB. The site‐specific incorporation of the azide affords control over protein orientation, thereby resulting in a homogeneous immobilization on the chip surface. This method can be used to study topology‐dependent interactions of any (membrane) protein.

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch

References and Relationships

• Bibliographic citation: Site‐Specific Immobilization of the Peptidoglycan Synthase PBP1B on a Surface Plasmon Resonance Chip Surface ; volume:17 ; number:23 ; year:2016 ; pages:2250-2256 ; extent:7 ; ChemBioChem ; 17, Heft 23 (2016), 2250-2256 (gesamt 7)

Contributors, Places and Time

• Creator: van't Veer, Inge L. ; Leloup, Nadia O. L. ; Egan, Alexander J. F. ; Janssen, Bert J. C. ; Martin, Nathaniel I. ; Vollmer, Waldemar ; Breukink, Eefjan

Further information

• DOI: 10.1002/cbic.201600461
• URN: urn:nbn:de:101:1-2022110704524315049824
• Rights: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 15.08.2025, 7:32 AM CEST