Intrinsically Unstructured Proteins - Experimental Identification of Intrinsically Unstructured Proteins

Experimental Identification of Intrinsically Unstructured Proteins

Intrinsically unfolded proteins, once purified, can be identified by various experimental methods. Folded proteins have a high density (partial specific volume of 0.72-0.74 mL/g) and commensurately small radius of gyration. Hence, unfolded proteins can be detected by methods that are sensitive to molecular size, density or hydrodynamic drag, such as size exclusion chromatography, analytical ultracentrifugation, Small angle X-ray scattering (SAXS), and measurements of the diffusion constant. Unfolded proteins are also characterized by their lack of secondary structure, as assessed by far-UV (170-250 nm) circular dichroism (esp. a pronounced minimum at ~200 nm) or infrared spectroscopy.

Unfolded proteins have exposed backbone peptide groups exposed to solvent, so that they are readily cleaved by proteases, undergo rapid hydrogen-deuterium exchange and exhibit a small dispersion (<1 ppm) in their 1H amide chemical shifts as measured by NMR. (Folded proteins typically show dispersions as large as 5 ppm for the amide protons.)

The primary method to obtain information on disordered regions of a protein is NMR spectroscopy. The lack of electron density in X-ray crystallographic studies may also be a sign of disorder.

Recently, new methods including Fast parallel proteolysis (FASTpp) have been introduced, which allow to determine the fraction folded/disordered without the need for purification.