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Enzyme–ligand interaction monitored by synchrotron radiation circular dichroism

DOI: 10.1007/978-1-0716-0163-1_6 DOI Help

Authors: Rohanah Hussain (Diamond Light Source) , Charlotte S. Hughes (Diamond Light Source) , Giuliano Siligardi (Diamond Light Source)
Co-authored by industrial partner: No

Type: Book Chapter
ISBN: 978-1-0716-0163-1
Book Chapter: 6

State: Published (Approved)
Published: November 2019
Diamond Proposal Number(s): 12182 , 14484 , 16778 , 19680

Open Access Open Access

Abstract: 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. 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 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 to measure 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.

Journal Keywords: Circular dichroism; Ligand binding; Titration; Binding constant; UV denaturation; Protein stability; Data processing

Subject Areas: Technique Development, Biology and Bio-materials

Instruments: B23-Circular Dichroism