| Summary: | IA<sub>3</sub> is an intrinsically disordered protein (IDP) that becomes helical when bound to yeast proteinase A (YPRA) or in the presence of the secondary stabilizer 2,2,2-trifluoroethanol (TFE). Here, site-directed spin-labeling (SDSL) continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy and circular dichroism (CD) are used to characterize the TFE-induced helical conformation of IA<sub>3</sub> for a series of spin-labeled cysteine scanning constructs and varied amino acid substitutions. Results demonstrate that the N-terminal concave helical surface of IA<sub>3</sub>, which is the buried interface when bound to YPRA, can be destabilized by the spin-label or bulky amino acid substitutions. In contrast, the helical tendency of IA<sub>3</sub> is enhanced when spin-labels are incorporated into the convex, i.e., solvent exposed, surface of IA<sub>3</sub>. No impact of the spin-label within the C-terminal region was observed. This work further demonstrates the utility and sensitivity of SDSL CW-EPR for studies of IDPs. In general, care must be taken to ensure the spin-label does not interfere with native helical tendencies and these studies provide us with knowledge of where to incorporate spin-labels for future SDSL investigations of IA<sub>3</sub>.
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