b subtilis phor periplasmic domain  (ATCC)

Journal: The Journal of Biological Chemistry

Article Title: The VanS sensor histidine kinase from type-B vancomycin-resistant enterococci recognizes vancomycin directly

doi: 10.1016/j.jbc.2025.110276

Figure Lengend Snippet: Reconstitution of VanS B into nanodiscs (NDs) . A , schematic representation of the typical VanS architecture. Two transmembrane helices (TM1 and TM2) flank a periplasmic sensor domain, the length of which varies significantly among different VanS proteins. The cytoplasmic portion of the protein contains a membrane-proximal region, which typically forms a HAMP domain, followed by the DHp (dimerization and histidine phosphotransfer) and CA (catalytic and ATP-binding) domains. B , for ND formation, detergent-solubilized protein is incubated with lipids and scaffolding protein, after which detergent is removed, triggering the spontaneous formation of protein-belted lipid discs around the hydrophobic face of the protein. The protein model shown represents the structure of full-length VanS B , as predicted by AlphaFold; domain colors match those found in panel (A) . C , size-exclusion chromatograms reveal that NDs containing VanS B elute earlier than empty NDs formed under identical conditions, indicating size increases consistent with the incorporation of the VanS protein. D , coomassie-stained denaturing SDS-PAGE gels showing a purified VanS B ND preparation indicating that this preparation contains approximately similar amounts of VanS B and the scaffolding protein MSP1D1.

Article Snippet: A similar strategy was used to produce tandem and single-copy constructs of the B. subtilis PhoR periplasmic domain (Uniprot P23545 aa 32–150), amplifying the appropriate region from B. subtilis subsp. subtilis strain 168 (American Type Culture Collection cat. no. 23857).

Techniques: Membrane, Binding Assay, Incubation, Scaffolding, Staining, SDS Page, Purification

Journal: The Journal of Biological Chemistry

Article Title: The VanS sensor histidine kinase from type-B vancomycin-resistant enterococci recognizes vancomycin directly

doi: 10.1016/j.jbc.2025.110276

Figure Lengend Snippet: The VanS B periplasmic sensor domain can be labeled with vancomycin-based photoprobes . A , the VanS B periplasmic sensor domain (residues 31–132) was expressed as a cleavable fusion with MBP, in both single-copy and tandem forms, and purified to homogeneity . B , two photoprobes were used, in which a photo-active diazirine was attached to either the antibiotic’s vancosamine sugar (Photoprobe V) or its N terminus (Photoprobe N). C , mass spectrometric evidence that both photoprobes label the purified tandem sensor-domain construct. D , vancomycin competes with both photoprobes for binding to the tandem sensor domain. Labeling efficiency was calculated from the mass spectrometric data, with efficiency expressed as the relative peak height for the photolabeled species as compared to that of the unlabeled species. E , confirmation of photolabeling using an anti-vancomycin Western-blot assay, showing labeling of both the isolated single-copy and tandem sensor domains, as well as full-length VanS B . Coomassie-stained gels serve as loading controls. Photoprobe N typically labels VanS B constructs less efficiently than Photoprobe V, as judged by both Western blotting and mass spectrometry (see panel (C) ); this may reflect differences in the diazirine group’s proximity to the protein, as determined by the conformation of the bound antibiotic.

Article Snippet: A similar strategy was used to produce tandem and single-copy constructs of the B. subtilis PhoR periplasmic domain (Uniprot P23545 aa 32–150), amplifying the appropriate region from B. subtilis subsp. subtilis strain 168 (American Type Culture Collection cat. no. 23857).

Techniques: Labeling, Purification, Construct, Binding Assay, Western Blot, Isolation, Staining, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: The VanS sensor histidine kinase from type-B vancomycin-resistant enterococci recognizes vancomycin directly

doi: 10.1016/j.jbc.2025.110276

Figure Lengend Snippet: Vancomycin binds directly to the periplasmic sensor domain of VanS B . A , fluorescently labeled vancomycin derivative used for fluorescence anisotropy measurements. B , BODIPY-FL-vancomycin binds to both the single and tandem sensor-domain constructs of VanS B . In contrast, single and tandem constructs of the PhoR negative control fail to bind the antibiotic. C and D , isothermal titration calorimetry confirms vancomycin binding by the single-copy (C) and tandem (D) VanS B sensor-domain constructs. The related glycopeptide antibiotic teicoplanin fails to bind to the sensor domain , which is consistent with the known behavior of type-B VRE. VRE, vancomycin-resistant enterococci.

Article Snippet: A similar strategy was used to produce tandem and single-copy constructs of the B. subtilis PhoR periplasmic domain (Uniprot P23545 aa 32–150), amplifying the appropriate region from B. subtilis subsp. subtilis strain 168 (American Type Culture Collection cat. no. 23857).

Techniques: Labeling, Fluorescence, Construct, Negative Control, Isothermal Titration Calorimetry, Binding Assay, Glycoproteomics