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Characterizing the Residue Level Folding of the Intrinsically Unstructured IA3

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Department of Biochemistry & Molecular Biology, University of Florida, Gainesville, Florida 32610-0245, Center for Medical, Agricultural, and Veterinary Entomology, USDA/ARS, Gainesville, Florida 32608, McKnight Brain Institute and National High Magnetic Field Laboratory, University of Florida, Gainesville, Florida 32610, and Department of Physics, University of Florida, Gainesville, Florida 32611-8440
Cite this: Biochemistry 2006, 45, 45, 13585–13596
Publication Date (Web):October 20, 2006
Copyright © 2006 American Chemical Society

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    Residue level analysis of the folding of simple proteins may hold the key to understanding folding pathways and aid in structure prediction. IA3, the endogenous inhibitor of yeast aspartic proteinase A (YPrA), is an unstructured protein in solution. Comparison of the 2D 15N-HSQC spectra of IA3 in water and in 23% 2,2,2-trifluoroethanol (TFE) shows that the individual residue cross peaks of IA3 become more dispersed in the presence of TFE, indicating that the protein undergoes an unstructured to structured transition in the presence of TFE. This transition can be monitored by the movements of the cross peaks. Following the individual cross peaks, however, is complicated and does not establish whether a single transition occurs globally in the sequence. In this equilibrium study, we apply singular value decomposition (SVD) to elucidate both the main features of the TFE-driven transition and the residue-level deviations from the average behavior. This analysis has yielded a two-state folding description as well as specifics of NMR frequency shifts of individual residues, indicating that the N-terminus of IA3 has a higher helical propensity than the C-terminus. Additionally, we discuss possible mechanisms for observed deviations from a two-state folding transition. When combined with a traditional biochemical understanding of interactions between individual residues, this approach leads to a better understanding of protein folding.

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     S.J.H. acknowledges the support of the National Science Foundation, MCB #0347124. Additional support was provided by NIH research Grant NCRR P41 RR016105.

     Department of Biochemistry & Molecular Biology, University of Florida.


     Center for Medical, Agricultural, and Veterinary Entomology, USDA/ARS.

     McKnight Brain Institute and National High Magnetic Field Laboratory, University of Florida.


     Address correspondence to Dr. Stephen J. Hagen, Department of Physics, University of Florida, P.O. Box 118440, Gainesville, Florida 32611-8440. Phone, 352-392-4716; fax, 352-392-7709; e-mail, [email protected].

     Department of Physics, University of Florida.

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    Figure depicting the 40 tracked chemical shifts of the IA3 sequence, and triple resonance data used to identify and assign individual peaks. This material is available free of charge via the Internet at

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    Cited By

    This article is cited by 14 publications.

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    3. Matthew Sweede, Gayatri Ankem, Boonta Chutvirasakul, Hugo F. Azurmendi, Souhad Chbeir, Justin Watkins, Richard F. Helm, Carla V. Finkielstein and Daniel G. S. Capelluto. Structural and Membrane Binding Properties of the Prickle PET Domain. Biochemistry 2008, 47 (51) , 13524-13536.
    4. Ranjani Narayanan, Omjoy K. Ganesh, Arthur S. Edison and Stephen J. Hagen . Kinetics of Folding and Binding of an Intrinsically Disordered Protein: The Inhibitor of Yeast Aspartic Proteinase YPrA. Journal of the American Chemical Society 2008, 130 (34) , 11477-11485.
    5. 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 2018, 5 (3) , 166-181.
    6. Thomas M. Casey, Zhanglong Liu, Jackie M. Esquiaqui, Natasha L. Pirman, Eugene Milshteyn, Gail E. Fanucci. Continuous wave W- and D-Band EPR spectroscopy offer “sweet-spots” for characterizing conformational changes and dynamics in intrinsically disordered proteins. Biochemical and Biophysical Research Communications 2014, 450 (1) , 723-728.
    7. Isabelle H. Barrette-Ng, Sau-Ching Wu, Wai-Mui Tjia, Sui-Lam Wong, Kenneth K. S. Ng. The structure of the SBP-Tag–streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits. Acta Crystallographica Section D Biological Crystallography 2013, 69 (5) , 879-887.
    8. Jakob R. Winther, Helen Webb, John Kay. Saccharopepsin. 2013, 128-133.
    9. Jin Wang, Yong Wang, Xiakun Chu, Stephen J. Hagen, Wei Han, Erkang Wang, . Multi-Scaled Explorations of Binding-Induced Folding of Intrinsically Disordered Protein Inhibitor IA3 to its Target Enzyme. PLoS Computational Biology 2011, 7 (4) , e1001118.
    10. Natasha L. Pirman, Eugene Milshteyn, Luis Galiano, Justin C. Hewlett, Gail E. Fanucci. Characterization of the disordered‐to‐α‐helical transition of IA 3 by SDSL‐EPR spectroscopy. Protein Science 2011, 20 (1) , 150-159.
    11. Piotr Kaczka, Agnieszka Polkowska‐Nowakowska, Krystyna Bolewska, Igor Zhukov, Jarosław Poznański, Kazimierz L. Wierzchowski. Backbone dynamics of TFE‐induced native‐like fold of region 4 of Escherichia coli RNA polymerase σ 70 subunit. Proteins: Structure, Function, and Bioinformatics 2010, 78 (3) , 754-768.
    12. . References. 2009, 265-312.
    13. Mariapina D’Onofrio, Laura Ragona, Dimitrios Fessas, Marco Signorelli, Raffaella Ugolini, Massimo Pedò, Michael Assfalg, Henriette Molinari. NMR unfolding studies on a liver bile acid binding protein reveal a global two-state unfolding and localized singular behaviors. Archives of Biochemistry and Biophysics 2009, 481 (1) , 21-29.
    14. Tim J. Winterburn, Lowri H. Phylip, Daniel Bur, David M. Wyatt, Colin Berry, John Kay. N‐terminal extension of the yeast IA 3 aspartic proteinase inhibitor relaxes the strict intrinsic selectivity. The FEBS Journal 2007, 274 (14) , 3685-3694.

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