UV Denaturation of Proteins Monitored by Circular Dichroism in the Far-UV Region (180–260 nm)
Rohanah Hussain, Charlotte S. Hughes, Giuliano Siligardi
Circular dichroism
Ligand binding
Titration
Binding constant
UV denaturation
Protein stability
Data processing
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 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.
Attachments
Steps
The general protocol is as follows:
For a cuvette (either cylindrical or rectangular cell) of 0.02 cm pathlength, 40µL at about 0.4mg/mL is loaded into the cuvette.
Standard data collection parameters in the far-UV region (180–260 nm) include 1 nm increments, 1 s integration time, and 1.2 nm bandwidth. At this point a standard CD measurement can be collected.
For the UV denaturation method, repeated continuous measurements are scanned. The number of scans to be collected is dependent on the system under study as the protein sensitive to UV light (photostability) varies from protein to protein. A suggested range is 20–30 scans that correspond to a total time of 1h 0m 0s–1h 30m 0s in order to obtain a good denaturation rate trend within a reasonable time-scale which is often a key consideration especially for Users of the B23 beamline at Diamond Light Source where allocated experimental time is limited.
For data analyses see part 3.4 "Processing of Circular Dichroism Data Collected at B23".
