|Organization||University of Florida|
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
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.