TITLE: Low-Volume Titrations for Ligand Binding Monitored by Circular Dichroism
AUTHORS: Rohanah  Hussain, Charlotte S. Hughes, Giuliano  Siligardi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div><br/><div class = "text-block">This chapter provides an overview of how to perform circular dichroism (CD) experiments, detailing methods, hints and tips for successful CD measurements. Descriptions of different experimental designs are discussed using CD to investigate ligand-binding interactions. This includes standard qualitative CD measurements conducted in both single-measurement mode and high-throughput 96-well plate mode, CD titrations, and UV protein denaturation assays with and without ligand.</div><br/><div class = "text-block">The highly collimated micro-beam available at B23 beamline for synchrotron radiation circular dichroism (SRCD) at Diamond Light Source (DLS) offers many advantages to benchtop instruments. The synchrotron light source is ten times brighter than a standard xenon arc light source of benchtop instruments. The small diameter of the synchrotron beam can be up to 160 times smaller than that of benchtop light beams; this has enabled the use of small aperture cuvette cells and flat capillary tubes reducing substantially the amount of volume sample to be investigated. Methods, hints and tips, and golden rules tomeasure good quality, artifact-free SRCD and CD data will be described in this chapter in particular for the study of protein–ligand interactions and protein photostability.</div></div>

[STEPS]
?. Measure the concentration of protein solution at A280. Use this value to calculate the dilution factor for the stock solution for the working solution. The concentration of the protein to be used during the experiment is in part determined with respect to the UV absorbance contribution of the ligand at each titration point which should not be more than 1.5–1.6 (see Notes 9–11).
?. Aliquots of ligand are added to the host mixture in the cuvette, pipetting up and down to thoroughly combine the components (see Notes 12 and 13).
?. Mixture incubated for a user-defined period of time (e.g., ) to allow for equilibration to occur (see Note 14) and ensure the appearance of the solution is homogenous before measuring (see Notes 13, 15 and 16). This is particularly important in the presence of detergents as the addition of aliquots affects the vesicle morphology and requires equilibration to obtain reproducible CD baselines. The way to determine the incubation time is to scan repeated consecutive CD spectra until the spectral shape is stable. The incubation time can be calculated as the product of the number of scans multiplied by the time to scan a single spectrum.
?. Measurements are collected for each titration point, after the determined incubation time.
?. CDApps is encouraged for the analysis, using the experimental plan as the template for the analysis as it contains information regarding volumes, concentration, and dilution factors of the mixture components in addition to pathlength(s) used to monitor the mixtures (see Notes 10–12).
?. CD spectrum of the Buffer under the same incubation time is measured to be used as baseline to be subtracted from each titration point spectrum, followed by the CD spectrum of the relevant proportion of the maximum-titrate ligand concentration. This is essential if the ligand is chiral. For achiral ligands, this is not necessary as the ligand will be devoid of any CD and the CD spectrum of the baseline will be sufficient for the data processing.
?. Difference in the CD signal at each point is plotted against the respective ligand concentration, fitting with a Hill function for determination of the binding dissociation constant Kd and the stoichiometry of the interaction using a nonlinear regression analysis [46].