Intrinsic disorder is a common feature in proteins yet is not completely understood. Analysis of the folding of simple proteins may hold the key to understanding the benefits of intrinsic disorder. IA₃ is a 68 amino acid endogenous inhibitor of yeast aspartic proteinase A (YPrA). In solution IA₃ can be found as an unstructured protein; however in the presence of YPrA, the N-terminus of IA₃ can be found as a helical structure bound to YPrA. This transition can be induced by the addition of the alcohol cosolvent 2,2,2-trifluoroethanol (TFE).

Circular dichroism studies of IA₃ in the presence of TFE indicated that the refolding midpoint between the coil and helix state is 16.3% TFE. However, these studies cannot conclusively state which residues are involved in the folding. Comparison of the 2D ¹⁵N-HSQC spectra of IA ₃ in water and in 23% TFE indicated that the protein undergoes an unstructured to structured transition in the presence of TFE. The refolding midpoint found by NMR is 18.3%, in agreement with the CD studies. The NMR studies allowed a residue level look how individual amino acids move toward a helical structure, with a greater helical tendency at the N-terminus.

Inhibition assays of YPrA with several IA₃ constructs show that only the N-terminal half of the protein is necessary to inhibit YPrA with the same specificity as the wild type protein. Isothermal titration calorimetry was employed to understand the function of the C-terminal portion of the molecule within its biological context. These studies demonstrated a marked difference in interactions between the wild type protein and the N-terminal half of IA ₃ with YPrA, suggesting that the C-terminus plays a role in the interaction between the two proteins apart from inhibition.

Assessment and comparison of the folding rates within a biological context and in TFE may shed light on the validity of the models used to understand such a simple system. Laser-induced temperature jump experiments were used to determine if the folding rate of IA₃ changes in the presence of YPrA.

This research demonstrates that the intrinsically disordered IA ₃ displays a rich set of interactions with YPrA. (Full text of this dissertation may be available via the University of Florida Libraries web site. Please check http://www.uflib.ufl.edu/etd.html)