Abstract
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. The difference CD spectra of the protein–ligand mixtures calculated subtracting the spectra of the ligand at various molar ratios can be used to determine the type of conformational changes induced by the ligand in terms of the estimated content of the various elements of protein secondary structure. The highly collimated microbeam and high photon flux of Diamond Light Source B23 beamline for synchrotron radiation circular dichroism (SRCD) enable the use of minimal amount of membrane proteins (7.5 µg for a 0.5 mg/ml solution) for high-throughput screening. Several examples of CD titrations of membrane proteins with a variety of ligands are described herein including the protocol tips that would guide the choice of the appropriate parameters to conduct these titrations by CD/SRCD in the best possible way.
Highlights
Membrane sensor kinaseTwo-component systems (TCSs) are important signalling systems present mostly in prokaryotic organisms and some lower eukaryotic organisms including the archae [1,2]
Apart from nuclear magnetic resonance (NMR), analytical ultracentrifugation (AUC) and isothermal titration calorimetry (ITC) do not provide direct information about the types of conformational changes induced by ligand binding and this is one of the main reasons why circular dichroism (CD)/synchrotron radiation circular dichroism (SRCD) should be used to determine directly the conformational behaviour of biologically important molecules as a function of environment such as temperature, pressure, pH, solvent composition, ionic strength, chemical agents, surfactants and ligandbinding interactions
A CD titration starts with the spectrum of the protein and subsequent spectra are scanned by adding small aliquots of ligand at incrementally increased molar ratios until the observed change reaches a saturation point
Summary
Membrane sensor kinaseTwo-component systems (TCSs) are important signalling systems present mostly in prokaryotic organisms and some lower eukaryotic organisms including the archae [1,2]. Apart from NMR, AUC and ITC do not provide direct information about the types of conformational changes induced by ligand binding and this is one of the main reasons why CD/SRCD should be used to determine directly the conformational behaviour of biologically important molecules as a function of environment such as temperature, pressure, pH, solvent composition, ionic strength, chemical agents, surfactants and ligandbinding interactions.
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