Spin-label scanning reveals conformational sensitivity of the bound helical interfaces of IA<sub>3</sub>
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...
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| Format: | Article |
| Language: | English |
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AIMS Press
2018-09-01
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| Series: | AIMS Biophysics |
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| Online Access: | http://www.aimspress.com/biophysics/article/3103/fulltext.html |
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| author | Katie M. Dunleavy Eugene Milshteyn Zachary Sorrentino Natasha L. Pirman Zhanglong Liu Matthew B. Chandler Peter W. D’Amore Gail E. Fanucci |
| author_facet | Katie M. Dunleavy Eugene Milshteyn Zachary Sorrentino Natasha L. Pirman Zhanglong Liu Matthew B. Chandler Peter W. D’Amore Gail E. Fanucci |
| author_sort | Katie M. Dunleavy |
| collection | DOAJ |
| description | 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>. |
| first_indexed | 2024-04-14T07:23:47Z |
| format | Article |
| id | doaj.art-b3f9fb6a4a5d4baa8cc77da8fd9e62ed |
| institution | Directory Open Access Journal |
| issn | 2377-9098 |
| language | English |
| last_indexed | 2024-04-14T07:23:47Z |
| publishDate | 2018-09-01 |
| publisher | AIMS Press |
| record_format | Article |
| series | AIMS Biophysics |
| spelling | doaj.art-b3f9fb6a4a5d4baa8cc77da8fd9e62ed2022-12-22T02:06:05ZengAIMS PressAIMS Biophysics2377-90982018-09-015316618110.3934/biophy.2018.3.166biophy-05-03-166Spin-label scanning reveals conformational sensitivity of the bound helical interfaces of IA<sub>3</sub>Katie M. DunleavyEugene MilshteynZachary SorrentinoNatasha L. PirmanZhanglong LiuMatthew B. ChandlerPeter W. D’AmoreGail E. Fanucci0Department of Chemistry, University of Florida, PO BOX 117200, Gainesville, FL 32611-7200, USAIA<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>.http://www.aimspress.com/biophysics/article/3103/fulltext.htmlsite-directed spin-labelingintrinsically disordered proteinIA<sub>3</sub>nitroxide labelelectron paramagnetic resonancecircular dichroism |
| spellingShingle | 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 IA<sub>3</sub> AIMS Biophysics site-directed spin-labeling intrinsically disordered protein IA<sub>3</sub> nitroxide label electron paramagnetic resonance circular dichroism |
| title | Spin-label scanning reveals conformational sensitivity of the bound helical interfaces of IA<sub>3</sub> |
| title_full | Spin-label scanning reveals conformational sensitivity of the bound helical interfaces of IA<sub>3</sub> |
| title_fullStr | Spin-label scanning reveals conformational sensitivity of the bound helical interfaces of IA<sub>3</sub> |
| title_full_unstemmed | Spin-label scanning reveals conformational sensitivity of the bound helical interfaces of IA<sub>3</sub> |
| title_short | Spin-label scanning reveals conformational sensitivity of the bound helical interfaces of IA<sub>3</sub> |
| title_sort | spin label scanning reveals conformational sensitivity of the bound helical interfaces of ia sub 3 sub |
| topic | site-directed spin-labeling intrinsically disordered protein IA<sub>3</sub> nitroxide label electron paramagnetic resonance circular dichroism |
| url | http://www.aimspress.com/biophysics/article/3103/fulltext.html |
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