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.

Z-ring membrane anchors associate with cell wall synthases to initiate bacterial cell division

“…This effect does not impair the quantification of other peaks (PG products). Tris-Tricine SDS-PAGE was used to separate glycan chains 70 , using the same protein concentrations and reaction conditions than in the TPase activity experiment at low PBP1A/PBP1B concentration but in the presence of 1 mM ampicillin to inhibit the TPase activity.…”

Induced conformational changes activate the peptidoglycan synthase PBP1B

“…In activity assays (either of the above) performed at reducing conditions, 10 mM TCEP (pH adjusted to 7.5 with NaOH) was added and proteins were incubated for 20 min at RT before proceeding. Measurement of glycan polymer lengths was performed largely as described previously with modifications to reaction conditions and the labelling of lipid II (Barrett et al, ; van’t Veer et al, ). Reactions were performed in a buffer of 20 mM HEPES/NaOH pH 7.5, 5 mM MgCl 2 , 150 mM NaCl, 1 mM ampicillin, 0.05% Triton X‐100 at 37°C.…”

Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins

“…When indicated ampicillin was added at mM and moenomycin was added at 50 µM. After plate reader measurements, reactions were stopped by boiling for 5 min, vacuumdried using a speed-vac desiccator and analysed by Tris-Tricine SDS-PAGE as previously described (Van't Veer et al, 2016).…”

“…Reactions contained 20 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 0.06% TX-100 and 1 mM Ampicillin to block transpeptidation. Aliquots were taken after 0, 2, 5, 10, 30 and 60 min incubation at 37°C, boiled for 10 min to stop reactions and analysed by Tris-Tricine SDS-PAGE followed by fluorescence detection as previously described (Van't Veer et al, 2016).…”