Circular Dichroism of Intrinsically Disordered Proteins

Abstract

10Circular dichroism (CD) in the far ultraviolet region is one of the most widely used methods for characterizing the secondary structure of proteins. Unordered polypeptides have a characteristic far -UV CD spectrum. Proteins that are disordered throughout their sequence are readily recognized by CD and CD has been used to identify and characterize a large number of IDPs. A common type of IDP in which a protein has folded domains interspersed with extensive unordered regions is more diffi cult to diagnose. Limited proteolysis can locate the boundaries between compact and unordered portions of the protein.Expression of the individual domains identifi ed by proteolysis, followed by CD analysis can be used to elucidate the structure of such proteins. INTRODUCTIONCircular dichroism (CD) in the far ultraviolet region is a widely used method for determining and monitoring the secondary structure of proteins (29,32,46) . α -Helices, β -sheets, β -turns, and unordered polypeptides give rise to characteristic CD spectra that can easily be recognized when one of these ABSTRACT conformations is dominant. Typical proteins contain most or all of these conformational types, but methods have been developed to analyze the CD spectrum of a protein and determine the fraction of residues in each conformation (5,8,28,41,47,49) . IDPs that lack any signifi cant organized secondary structure give the characteristic CD spectrum of an unordered polypeptide, with a strong negative band near 200 nm and either a weak negative shoulder or a weak positive maximum near 220 nm. Such IDPs are readily recognized by CD. It is more challenging to recognize and characterize IDPs that contain folded domains intermingled with extensive unfolded regions. Progress has recently been made with some specifi c IDPs by defi ning the domains through limited proteolysis, followed by cloning and expression of individual domains that can be analyzed by CD and other methods.In this chapter, the CD of unfolded proteins will be described, starting with model polypeptides and proceeding to globular proteins unfolded by various means. The CD of IDPs will then be reviewed, beginning with full -length IDPs and concluding with IDPs that have both ordered and unordered regions.This chapter considers only CD in the UV region, that is, electronic CD. CD in the infrared, vibrational CD (VCD), and the related phenomenon of Raman optical activity are discussed in chapter 11 by Reinhard

High‐order oligomers of intrinsically disordered brain proteins BASP1 and GAP‐43 preserve the structural disorder

“…The structure of a coil‐like IDP represents a statistical coil with a high contribution of PPII conformation . The CD ellipticity values at 200 and 222 nm indicated a high content (> 30%) of PPII helix in BASP1 and GAP‐43.…”

“…In a coil‐like IDP, the PPII conformation is a part of a statistical coil ensemble. Given that the PPII population is temperature dependent, the CD spectrum of a coil‐like IDP can be expressed as a superposition of two components: the low‐temperature PPII conformation and the high‐temperature truly unordered conformation (which is close to a random coil), both of which have spectral bands around 200 and 222 nm . In this context, the PPII fraction can be calculated according to the method of Park et al .…”

“…This estimation should be considered as a lower limit to the actual PPII content because the high‐temperature form ensemble also contains residues in PPII conformation .…”

Defining Structural Domains of an Intrinsically Disordered Protein: Sic1, the Cyclin-Dependent Kinase Inhibitor of Saccharomyces cerevisiae

“…The CD spectra of IDPs are dominated by the characteristic features of random-coil polypeptides, with a strong negative band around 200 nm and still negative ellipticity values at 190 nm [37]. The complementary fragments Sic1 D186 and Sic1 1-186 were analyzed by CD spectroscopy in the far UV, in order to probe their secondary-structure properties.…”

Insights into the coiled-coil organization of the Hendra virus phosphoprotein from combined biochemical and SAXS studies

“…S1C), again suggesting the lack of interacting helices through a quaternary structure organization. The lower than 1.0 value of Θ 222/208 probably reflects the contribution of disordered regions, which are typified by very low ellipticities values at 222 nm (Woody, 2010). Thermal denaturation experiments similar to those described above, yielded comparable results in that thermal unfolding was found to be reversible, thus ruling out possible heat-induced protein aggregation and arguing for proper refolding Table 1 SAXS data collection and scattering-derived structural parameters (at the highest concentration) for PMD and PCT.…”