Circular Dichroism Techniques for the Analysis of Intrinsically Disordered Proteins and Domains

Abstract

Circular dichroism (CD) spectroscopy is a simple and powerful technique, which allows for the assessment of the conformational properties of a protein or protein domain. Intrinsically disordered proteins (IDPs), as discussed throughout this series, differ from random coil polypeptides in that different regions present specific conformational preferences, exhibiting dynamic secondary structure content [1]. These dynamic secondary structure elements can be stabilized or perturbed by different chemical (solvent, ionic strength, pH) or physical (temperature) agents, by posttranslational modifications, and by ligands. This information is important for defining ID nature. As IDPs present dynamic conformations, circular dichroism measurements (and other approaches as well) should be carried out not as single spectra performed in unique conditions, but instead changing the chemical conditions and observing the behavior, as part of the determination of the ID nature.In this chapter, we present the basic methodology for performing Far-UV CD measurements on a protein of interest and for identifying and characterizing intrinsically disordered regions, and several protocols for the analysis of residual secondary structure present in the protein under study. These techniques are straightforward to perform; they require minimal training and can be preliminary to more complex methodologies such as NMR.

Characterization of the Grp94/OS-9 Chaperone–Lectin Complex

“…While well-folded proteins classically denature with a cooperative transition, proteins containing a high proportion of unstructured polypeptide exhibit non-cooperative transitions. 40 A thermal denaturation scan for OS-9 ΔMRH showed a linear decrease in ellipticity at 222 nm with increasing temperature (Figure 7B), ultimately reaching a plateau at 85 °C, suggesting that the C-terminal domain of OS-9 is intrinsically disordered. A similar CD spectral analysis of the isolated OS-9 CTE, a major in vitro Grp94 binding site, showed minima at 208 and 222 nm suggesting a predominantly helically structured peptide (Figure 7C).…”

“…While the band at 222 nm derives from α-helical structure, the band at 204 nm indicates the presence of intrinsically disordered regions and arises from the combination of two minima, one at 200 nm arising from random coil and the other at 208 nm arising from α-helical content. 40; 41 …”

The Proline/Glycine-Rich Region of the Biofilm Adhesion Protein Aap Forms an Extended Stalk that Resists Compaction

“…Comparison of their ability to block hydrogen exchange revealed that unlike urea, GdnHCl does not form H-bonds with the polypeptide but instead stacks against planar groups in the backbone and side chains such as Asn or aromatic residues [86]. Both urea and GdnHCl favor formation of the highly extended PPII conformation in IDPs and denatured globular proteins, monitored by the far-UV CD signal near 220 nm [44, 68, 87, 88]. PGR shows an increase in PPII content at high concentrations of urea (Fig.…”

Phosphorylation regulates the secondary structure and function of dentin phosphoprotein peptides

“…A cell path length of 1 mm was used, with protein concentrations between 10–20 mg/ml (4–6 uM). Data was converted to mean residue molar ellipticity ([θ] in deg cm 2 dmol-1) [equation 1] and the percent helicity [equation 2] was estimated using [θ]222 [27, 49]. $$\left[\theta \right]=\left(100\theta \right)∕\left(\text{clN}\right)$$ Where θ is the measured ellipticity (mdeg), c is the sample concentration (mM), 1 is the path length (cm), and N is the number of amino acids.…”

“…(i) Small-angle X-ray scattering (SAXS)/Wide-angle X-ray scattering (WAXS), which has been previously used to characterize highly flexible systems in solutions including disordered proteins [25] and have the additional advantage to provide information about molecular size, shape, aggregation, conformation changes [26]. (ii) Circular dichroism (CD), widely used to describe the secondary structure of proteins in solution [27]. (iii) Fourier transform infrared spectroscopy (FTIR) that provides information on the secondary structure of proteins not only in solution but also when they interact with solid surfaces or soluble molecular partners [28].…”