Uncovering Order within the Disorder: Redefining IA3's Intrinsically Disordered Properties

Abstract

IA3 is a 68 residue intrinsically disordered protein (IDP), found in Saccharomyces cerevisiae, that adopts N-terminal α-helical secondary structure when bound to yeast proteinase A (YPRA). The α-helical transition of IA3 can be stabilized in the absence of YPRA, by using 2,2,2-trifluoroethanol (TFE). Circular dichroism spectroscopy (CD) was used to characterize the unstructured-to-structured transition of wild type IA3 along with residue substituted mutants of this protein. These mutational studies showed TFE induced impacts on helical intensity in the structured state, but also uncovered variations in the unstructured, or disordered state of the protein. The observed structural differences in the presence of TFE indicated changes to helical intensity, however, we also found differences in residual order in the unfolded states, in the absence of TFE. Residual order of an IDP in an unfolded state suggests a distinction between a disordered and denatured protein, where residual order can be removed, an idea of “order within the disorder”. We found that the addition of differing amounts of urea removes the residual structure where wild type and all variants converge to a similar degree of un-folding, indicating a thermodynamic difference in the order of these unstructured ensembles. Dynamic light scattering (DLS) along with differential scanning calorimetry (DSC) results will be presented to further analyze changes in the disordered structure through thermal melting, and urea-induced denaturation. DLS and DSC results will characterize the changes in hydrodynamic radius, along with a determined melting temperature, the enthalpy of unfolding, and heat capacity change upon denaturation of the disordered state. These combined techniques will probe the presence of an energy barrier in the disordered state of IA3 that could indicate an ability to disrupt the “order within the disorder”.