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

Autor: Dunleavy, Katie M., Milshteyn, Eugene, Sorrentino, Zachary, Pirman, Natasha L., Liu, Zhanglong, Chandler, Matthew B., D'Amore, Peter W., Fanucci, Gail E.
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Zdroj: AIMS Biophysics; 2018, Vol. 5 Issue 3, p166-181, 16p
Abstrakt: 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. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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