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.
Highlights
Circular dichroism (CD) is a powerful technique which enables the monitoring of local and global changes in the structure andThe original version of this chapter was revised
CD spectroscopy enables the selective monitoring of specific chromophores of the protein including peptide backbone in the far-UV region (180–250 nm), aromatic side chains of amino acid residues and dihedral angles of disulfide bonds in the near-UV region (250–350 nm), and prosthetic groups in the visible region (400–800 nm)
Synchrotron radiation circular dichroism (SRCD) beamlines utilize the light produced at synchrotrons as the light source that is brighter than standard xenon light sources [1], and with much higher photon flux in the vacuum-UV region (130–200 nm)
Summary
Circular dichroism (CD) is a powerful technique which enables the monitoring of local and global changes in the structure and. CD spectroscopy enables the selective monitoring of specific chromophores of the protein including peptide backbone in the far-UV region (180–250 nm), aromatic side chains of amino acid residues and dihedral angles of disulfide bonds in the near-UV region (250–350 nm), and prosthetic groups (hemes, flavones, carotenoids) in the visible region (400–800 nm). The amide bond is the main chromophore that absorbs in the far-UV region (180–250 nm) [18], while the aromatic side chains of tryptophan, tyrosine, and phenylalanine amino acid residues and disulfide bonds are the chromophores that absorbs in the near-UV region Prosthetic groups such as hemes, NAD and FAD cofactors, and carotenoid pigments are the chromophores that extend the absorption of light in the visible region (400–800 nm). The CD/SRCD in the near-UV region is sensitive to the local environment of the aromatic side chains of Trp, Tyr, and Phe, and dihedral angle of disulfide bonds of cystine residues and has been used successfully to probe qualitatively and quantitatively ligand-binding interactions [25]
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