Two-component signal transduction systems are the main mechanism by which bacteria sense and respond to their environment, and their membrane-located histidine protein kinases generally constitute the sensory components of these systems. Relatively little is known about their fundamental mechanisms and precise nature of the molecular signals sensed, because of the technical challenges of producing sufficient quantities of these hydrophobic membrane proteins. This study evaluated the heterologous production, purification and activities of the 16 intact membrane sensor kinases of Enterococcus faecalis. Following the cloning of the genes into expression plasmid pTTQ18His, all but one kinase was expressed successfully in Escherichia coli inner membranes. Purification of the hexa-histidine 'tagged' recombinant proteins was achieved for 13, and all but one were verified as intact. Thirteen intact kinases possessed autophosphorylation activity with no added signal when assayed in membrane vesicles or as purified proteins. Signal testing of two functionally-characterized kinases, FsrC and VicK, was successful examplifying the potential use of in vitro activity assays of intact proteins for systematic signal identification. Intact FsrC exhibited an approximately 10-fold increase in activity in response to a two-fold molar excess of synthetic GBAP pheromone, whilst glutathione, and possibly redox potential, were identified for the first time as direct modulators of VicK activity in vitro. The impact of DTT on VicK phosphorylation resulted in increased levels of phosphorylated VicR, the downstream response regulator, thereby confirming the potential of this in vitro approach for investigations of modulator effects on the entire signal transduction process of two-component systems.
VanA-type resistance to glycopeptide antibiotics in clinical enterococci is regulated by the VanSARA two-component signal transduction system. The nature of the molecular ligand that is recognised by the VanSA sensory component has not hitherto been identified. Here we employ purified, intact and active VanSA membrane protein (henceforth referred to as VanS) in analytical ultracentrifugation experiments to study VanS oligomeric state and conformation in the absence and presence of vancomycin. A combination of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge (SEDFIT, SEDFIT-MSTAR and MULTISIG analysis) showed that VanS in the absence of the ligand is almost entirely monomeric (molar mass M = 45.7 kDa) in dilute aqueous solution with a trace amount of high molar mass material (M ~ 200 kDa). The sedimentation coefficient s suggests the monomer adopts an extended conformation in aqueous solution with an equivalent aspect ratio of ~(12 ± 2). In the presence of vancomycin over a 33% increase in the sedimentation coefficient is observed with the appearance of additional higher s components, demonstrating an interaction, an observation consistent with our circular dichroism measurements. The two possible causes of this increase in s – either a ligand induced dimerization and/or compaction of the monomer are considered.
FsrC is the membrane-bound histidine kinase component of the Fsr two-component signal transduction system involved in quorum sensing in the hospital-acquired infection agent Enterococcus faecalis. Synchrotron radiation circular dichroism spectroscopy was used here to study the intact purified protein solubilised in detergent micelles. Conditions required for FsrC stability in detergent were firstly determined and tested by prolonged exposure of stabilised protein to far-ultraviolet radiation. Using stabilised purified protein, far-ultraviolet synchrotron radiation circular dichroism revealed that FsrC is 61% alpha-helical and that it is relatively thermostable, retaining at least 57% secondary structural integrity at 90 degrees C in the presence or absence of gelatinase biosynthesis-activating pheromone (GBAP). Whilst binding of the quorum pheromone ligand GBAP did not significantly affect FsrC secondary structure, near-ultraviolet spectra revealed that the tertiary structure in the regions of the Tyr and Trp residues was significantly affected. Titration experiments revealed a calculated kd value of 2 microM indicative of relatively loose binding ofgelatinase biosynthesis-activating pheromone to FsrC. Although use of synchrotron radiation circular dichroism has been applied to membrane proteins previously, to our knowledge this is the first report of its use to determine a kd value for an intact membrane protein. Based on our findings, we suggest that synchrotron radiation circular dichroism will be a valuable technique for characterising ligand binding by other membrane sensor kinases and indeed other membrane proteins in general. It further provides a valuable screening tool for membrane protein stability under a range of detergent conditions prior to downstream structural methods such as crystallisation and NMR experiments particularly when lower detergent concentrations are used.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2025 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.