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 disulphide 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-UV 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. 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. The highly collimated microbeam 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 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 to measure good quality, artifact-free SRCD and CD data will be described in this chapter in particular for the study protein-ligand interactions and protein photostability.