Spin-label scanning reveals conformational sensitivity of the bound helical interfaces of IA<sub>3</sub>

Katie M. Dunleavy; Eugene Milshteyn; Zachary Sorrentino; Natasha L. Pirman; Zhanglong Liu; Matthew B. Chandler; Peter W. D’Amore; Gail E. Fanucci; Katie M. Dunleavy; Eugene Milshteyn; Zachary Sorrentino; Natasha L. Pirman; Zhanglong Liu; Matthew B. Chandler; Peter W. D’Amore; Gail E. Fanucci

doi:10.3934/biophy.2018.3.166

AIMS Biophysics

2018, Volume 5, Issue 3: 166-181. doi: 10.3934/biophy.2018.3.166

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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

Received: 01 June 2018 Accepted: 01 August 2018 Published: 25 September 2018

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.
- site-directed spin-labeling,
- intrinsically disordered protein,
- IA₃,
- nitroxide label,
- electron paramagnetic resonance,
- circular dichroism
Citation: Katie M. Dunleavy, Eugene Milshteyn, Zachary Sorrentino, Natasha L. Pirman, Zhanglong Liu, Matthew B. Chandler, Peter W. D’Amore, Gail E. Fanucci. Spin-label scanning reveals conformational sensitivity of the bound helical interfaces of IA3[J]. AIMS Biophysics, 2018, 5(3): 166-181. doi: 10.3934/biophy.2018.3.166

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Abstract

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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