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Identifying Disordered Regions in Proteins by Limited Proteolysis

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Intrinsically Disordered Protein Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 896))

Abstract

Limited proteolysis experiments can be successfully used to detect sites of disorder in otherwise folded globular proteins. The approach relies on the fact that the proteolysis of a polypeptide substrate requires its binding in an extended conformation at the protease’s active site and thus an enhanced backbone flexibility or local unfolding of the site of proteolytic attack. A striking correlation was found between sites of limited proteolysis and sites of enhanced chain flexibility of the polypeptide chain, this last evaluated by the crystallographically determined B-factor. In numerous cases, it has been shown that limited proteolysis occurs at chain regions characterized by missing electron density and thus being disordered. Therefore, limited proteolysis is a simple and reliable experimental technique that can detect sites of disorder in proteins, thus complementing the results that can be obtained by the use of other physicochemical and computational approaches.

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Acknowledgments

This work was supported by the University of Padua (ex-60%) and by the Italian Ministry of University and Research (PRIN-2007). The former lab members Paola Picotti and Marcello Zambonin have contributed a great deal to the ideas herewith expressed.

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Authors and Affiliations

  1. CRIBI Biotechnology Centre, University of Padua, Padua, Italy

    Angelo Fontana, Patrizia Polverino de Laureto, Barbara Spolaore & Erica Frare

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  1. Angelo Fontana
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  2. Patrizia Polverino de Laureto
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  3. Barbara Spolaore
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  4. Erica Frare
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Correspondence to Angelo Fontana .

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  1. DEPT OF MOLECULAR MED, College of Medicine, University of South Florida, BRUCE B DOWNS BLVD 12901, TAMPA, 33612, Florida, USA

    Vladimir N. Uversky

  2. Dept. Biochemistry & Molecular Biology, Center for Computational Biology &, Indiana University, West 10th Street 410, Indianapolis, 46202, Indiana, USA

    A. Keith Dunker

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Fontana, A., de Laureto, P.P., Spolaore, B., Frare, E. (2012). Identifying Disordered Regions in Proteins by Limited Proteolysis. In: Uversky, V., Dunker, A. (eds) Intrinsically Disordered Protein Analysis. Methods in Molecular Biology, vol 896. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-3704-8_20

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  • DOI: https://doi.org/10.1007/978-1-4614-3704-8_20

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