Correction To: Enzyme-Ligand Interaction Monitored by Synchrotron Radiation Circular Dichroism.

The original version of the chapter Enzyme-Ligand Interaction Monitored by Synchrotron Radiation Circular Dichroism was previously published non-open access. It has now been changed to open access under a CC BY 4.0 license and the copyright holder has been updated to 'The Author(s).

Enzyme-Ligand Interaction Monitored by Synchrotron Radiation Circular Dichroism.

CD spectroscopy is the essential tool to quickly ascertain in the far-UV region the global conformational changes, the secondary structure content, and protein folding and in the near-UV region the local tertiary structure changes probed by the local environment of the aromatic side chains, prosthetic groups (hemes, flavones, carotenoids), the dihedral angle of disulfide bonds, and the ligand chromophore moieties, the latter occurring as a result of protein-ligand binding interaction. Qualitative and quantitative investigations into ligand-binding interactions in both the far- and near-UV regions using CD spectroscopy provide unique and direct information whether induced conformational changes upon ligand binding occur and of what nature that are unattainable with other techniques such as fluorescence, ITC, SPR, and AUC.This chapter provides an overview of how to perform circular dichroism (CD) experiments, detailing methods, hints and tips for successful CD measurements.

The gelatinase biosynthesis-activating pheromone binds and stabilises the FsrB membrane protein in Enterococcus faecalis quorum sensing.

Quorum-sensing mechanisms regulate gene expression in response to changing cell-population density detected through pheromones. In Enterococcus faecalis, Fsr quorum sensing produces and responds to the gelatinase biosynthesis-activating pheromone (GBAP). Here we establish that the enterococcal FsrB membrane protein has a direct role connected with GBAP by showing that GBAP binds to purified FsrB.

Characterisation of sensor kinase by CD spectroscopy: golden rules and tips.

This is a review that describes the golden rules and tips on how to characterise the molecular interactions of membrane sensor kinase proteins with ligands using mainly circular dichroism (CD) spectroscopy. CD spectroscopy is essential for this task as any conformational change observed in the far-UV (secondary structures (α-helix, β-strands, poly-proline of type II, β-turns, irregular and folding) and near-UV regions [local environment of the aromatic side-chains of amino acid residues (Phe, Tyr and Trp) and ligands (drugs) and prosthetic groups (porphyrins, cofactors and coenzymes (FMN, FAD, NAD))] upon ligand addition to the protein can be used to determine qualitatively and quantitatively ligand-binding interactions. Advantages of using CD versus other techniques will be discussed.

Spectroscopy data of ceftriaxone-lysozyme interaction and computational studies.

The data article presents the results obtained from fluorescence and synchrotron radiation circular dichroism spectroscopies about the lysozyme-ceftriaxone interaction at neutral and acidic pH values as well as the computational calculations described in the accompanying research article (Ruzza et al., sub) [1].

Chaperone-like effect of ceftriaxone on HEWL aggregation: A spectroscopic and computational study.

Background: Lysozyme is a widely distributed enzyme present in a variety of tissue and body fluids. Human and hen egg white lysozyme are used as validated model to study protein folding and stability and to understand protein misfolding and aggregation. We recently found that ceftriaxone, a β-lactam antibiotic able to overcome the blood-brain barrier, successfully eliminated the cellular toxic effects of misfolded proteins as Glial Fibrillary Acidic Protein (GFAP) and α-synuclein.

To Boil an Egg: Substrate Binding Affects Critical Stability in Thermal Unfolding of Proteins.

Thermal unfolding of proteins is used extensively in screening of drug candidates because molecular interactions with ligands and substrates affect strongly protein stability, transition temperature, and cooperativity. We use synchrotron radiation circular dichroism to monitor the thermal evolution of secondary structure in proteins as they approach the melting point and the impact of substrate on their thermal behavior. Using Landau free energy expansion, we quantify transition strength and proximity to a critical point through the relative separation τ between the transition temperature T and the spinodal T, obtained from the equation of state.

Hydrodynamics of the VanA-type VanS histidine kinase: an extended solution conformation and first evidence for interactions with vancomycin.

VanA-type resistance to glycopeptide antibiotics in clinical enterococci is regulated by the VanSR two-component signal transduction system. The nature of the molecular ligand that is recognised by the VanS sensory component has not hitherto been identified. Here we employ purified, intact and active VanS 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.

Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions.

This article reviews current methods for the reliable heterologous overexpression in Escherichia coli and purification of milligram quantities of bacterial membrane sensor kinase (MSK) proteins belonging to the two-component signal transduction family of integral membrane proteins. Many of these methods were developed at Leeds alongside Professor Steve Baldwin to whom this review is dedicated. It also reviews two biophysical methods that we have adapted successfully for studies of purified MSKs and other membrane proteins-synchrotron radiation circular dichroism (SRCD) spectroscopy and analytical ultracentrifugation (AUC), both of which are non-immobilization and matrix-free methods that require no labelling strategies.