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Spin-label scanning reveals conformational sensitivity of the bound helical interfaces of IA3

1 Department of Chemistry, University of Florida, PO BOX 117200, Gainesville, FL 32611-7200, USA
2 Department of Radiology and Biomedical Imaging, University of California, 505 Parnassus Avenue, San Francisco, CA 94143, USA
3 College of Medicine, University of Florida, 1600 SW Archer Road M509, Gainesville, FL 32610, USA
4 MilliporeSigma, 400 Summit Drive, Burlington, MA 01803, USA
5 CVS Health, 1 CVS Drive, Woonsocket, RI 02895, USA
6 Expertus Laboratories, Inc. 485 North US Highway 17 92 #415, Tampa, FL 32750, USA
7 Department of Orthopedic Surgery, LSU Health Science Center, 1542 Tulane Avenue, New Orleans, LA 70112, USA

† These three authors contributed equally.

Topical Section: Structural analysis of macromolecules

IA3 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 IA3 for a series of spin-labeled cysteine scanning constructs and varied amino acid substitutions. Results demonstrate that the N-terminal concave helical surface of IA3, 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 IA3 is enhanced when spin-labels are incorporated into the convex, i.e., solvent exposed, surface of IA3. 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 IA3.
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© 2018 the Author(s), licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution Licese (http://creativecommons.org/licenses/by/4.0)

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