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Limited Proteolysis of Protein-Nucleic Acid Complexes

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DNA-Protein Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 148))

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Abstract

Limited Proteolysis is a useful structural probe for investigating the globular nature of proteins by preferentially digesting the more accessible regions often found between domains. Generally, proteases require a small region of polypeptide chain possessing conformational flexibility for accommodation in the active site (1). The regions of a protein possessing conformational flexibility are often found between tightly folded domains and are, therefore, preferential sites for proteolysis. In practice, limited proteolysis is achieved by dilution of the enzyme sufficiently so that it will only digest the most accessible regions leaving the domains intact. Digestion of protein-nucleic acid is often advantageous in that the DNA may provide steric protection of the DNAbinding domain not afforded by the free protein. The generation of domains by limited proteolysis relies directly on the tertiary structure of the protein under investigation and provides much firmer evidence for their existence than that provided by sequence homology.

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References

  1. Vita, C., Dalzoppo, D., and Fontana, A (1987) Limited proteolysis of globular proteins: molecular aspects deduced from studies on thermolysin, in Macromolecular Biorecognition (Chaiken, I., Chaiancone, E., Fontana, A., and Veri, P., eds.), Humana Press, Clifton, NJ.

    Google Scholar 

  2. Merril, B., Stone, K., Cobianchi, F., Wilson, S., and Williams, K. (1988) Phenylalanines that are conserved among several RNA-binding proteins form part of a nucleic acid binding pocket in the heterogeneous nuclear ribonucleoprotein. J. Biol. Chem. 263, 3307–3313.

    Google Scholar 

  3. Bandziulis, R., Swanson, M., and Dreyfuss, G. (1989) RNA binding proteins as developmental regulators. Genes Dev. 4, 431–437.

    Article  Google Scholar 

  4. Plyte, S.E. and Kneale, G.G. (1993) Characterization of the DNA-binding domain of the Pf 1 gene 5 protein. Biochemistry 32, 3623–3628.

    Article  PubMed  CAS  Google Scholar 

  5. Huet, J., Conesa, C., Carles, C., and Sentenac, A. (1997) A cryptic DNA-binding domain at th COOH terminus of TFIIIB70 affects formation, stability and function of perinitiation complexes. J. Biol. Chem. 272, 18,341–18,349.

    Article  PubMed  CAS  Google Scholar 

  6. Bochareva, E., Frappier, L., Edwards, A., and Bochareva, A. (1998) The RPA32 subunit of human replication protein A contains a single stranded DNA-binding domain. J. Biol. Chem., 273, 3932–3936.

    Article  Google Scholar 

  7. Wigley, D., Davies, G., Dodson, E., Maxwell, A., and Dodson, G. (1991) Crystal structure of an N-terminal fragment of the DNA gyrase B protein. Nature 351, 624–629.

    Article  PubMed  CAS  Google Scholar 

  8. Kneale, G.G. (1983) Dissociation of the Pf1 nucleoprotein assembly complex and characterization of the DNA-binding protein. Biochem. Biophys. Acta 739, 216–224.

    PubMed  CAS  Google Scholar 

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© 2001 Humana Press Inc., Totowa, NJ

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Plyte, S.E., Geoff Kneale, G. (2001). Limited Proteolysis of Protein-Nucleic Acid Complexes. In: Moss, T. (eds) DNA-Protein Interactions. Methods in Molecular Biology, vol 148. Humana Press. https://doi.org/10.1385/1-59259-208-2:315

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  • DOI: https://doi.org/10.1385/1-59259-208-2:315

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-625-3

  • Online ISBN: 978-1-59259-208-1

  • eBook Packages: Springer Protocols

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