title: 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.
authors.affiliation: Department of Membrane Biochemistry and Biophysics, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
authors.forename: Inge L
authors.identifier: 0000-0003-3332-6894
authors.initials: IL
authors.lastname: Van't Veer
authors.affiliation: Crystal and Structural Chemistry, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
authors.forename: Nadia O L
authors.initials: NO
authors.lastname: Leloup
authors.affiliation: The Centre for Bacterial Cell Biology, Newcastle University, Richardson Road, NE2 4AX, Newcastle upon Tyne, UK.
authors.forename: Alexander J F
authors.initials: AJ
authors.lastname: Egan
authors.affiliation: Crystal and Structural Chemistry, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
authors.forename: Bert J C
authors.initials: BJ
authors.lastname: Janssen
authors.affiliation: Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG, Utrecht, The Netherlands.
authors.forename: Nathaniel I
authors.initials: NI
authors.lastname: Martin
authors.affiliation: The Centre for Bacterial Cell Biology, Newcastle University, Richardson Road, NE2 4AX, Newcastle upon Tyne, UK.
authors.forename: Waldemar
authors.initials: W
authors.lastname: Vollmer
authors.affiliation: Department of Membrane Biochemistry and Biophysics, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
authors.forename: Eefjan
authors.initials: E
authors.lastname: Breukink
mesh_terms: D001386:Azides / Q000737:chemistry / Q000378:metabolism; D034242:Cyclooctanes / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D046915:Penicillin-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D046928:Peptidoglycan Glycosyltransferase / Q000737:chemistry* / Q000378:metabolism; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000737:chemistry* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013499:Surface Properties
chemical_list: D001386:Azides; D034242:Cyclooctanes; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D055767:Immobilized Proteins; D046915:Penicillin-Binding Proteins; D046928:Peptidoglycan Glycosyltransferase; C478681:penicillin-binding protein 1B, E coli; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase
keywords: PBP; click chemistry; protein modifications; site specific immobilization; surface plasmon resonance