Spin-Labeling Electron Paramagnetic Resonance and Overhauser Dynamic Nuclear Polarization Characterizations of the Folding of IA₃, an Intrinsically Disordered Protein

Katie Dunleavy¹, Zachary Sorrentino², Eugene Milshteyn³, Thomas M. Casey⁴, Zhanglong Liu¹, and Gail E. Fanucci¹

1-Department of Chemistry, University of Florida , Gainesville, FL 32611
2-College of Medicine, University of Florida, Gainesville, FL 32610
3-Department of Bioengineering, University of California-Berkeley, Berkeley, California 94720
4-Bruker BioSpin Corporation, Billerica, MA 01821

IA₃ is an intrinsically disordered protein (IDP), found in Sacchoromyeces cerevisiae, that has been previously shown to adapt α-helical secondary structure when bound to yeast proteinase A (YPRA). The α-helical structure of IA₃ can be stabilized in the absence of YPRA, by using 2,2,2-trufluoroethanol (TFE). Site-directed spin-labeling (SDSL) along with electron paramagnetic resonance (EPR) has been used to characterize the TFE induced folding of IA₃. Here we report results from CYS scanning through the N- termini, which reveals evidence for the degree of unfoldedness of the region. Additionally, results demonstrate a sensitivity of the N-terminal region to amino acid substitution. Particularly site V8 is sensitive to mutation, where alterations in amino acid structure are shown to inhibit the TFE induced structural transition. We find that site I11C-SL behaves similarly to WT in TFE-induced folding, making site-11 a useful spin-label reporter site to probe the impact of mutations at val-8.  Results from circular dichroism, EPR and Overhauser dynamic nuclear polarization (ODNP) will be presented. ODNP is used to further understand hydration effects on protein folding. Hydration effects impact proper folding of proteins, and the study of IA₃’s hydration when disordered or ordered in structure, and mutants where folding is abbrogated can lead to a clearer representation of the secondary structure of this model IDP. ODNP will help us to understand the changes in this model system upon site directed mutation of amino acid residues to a cysteine, necessary for SDSL-EPR studies. Through the use of circular dichroism, SDSL-EPR, and ODNP, a clearer representation of the disordered and ordered states of IA₃ will be determined. This model system can further be used to hypothesize about other functional IDPs and their structural transition upon protein function.