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Distinct conformational stability and functional activity of four highly homologous endonuclease colicins

van den Bremer, E T J, Keeble, A H, Jiskoot, W, Spelbrink, R E J, Maier, C S, van Hoek, A, Visser, A J W G, James, R, Moore, G R, Kleanthous, C and Heck, A J R (2004) Distinct conformational stability and functional activity of four highly homologous endonuclease colicins. Protein Science. pp. 1391-1401. ISSN 1469-896X

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The family of conserved colicin DNases E2, E7, E8, and E9 are microbial toxins that kill bacteria through random degradation of the chromosomal DNA. In the present work, we compare side by side the conformational stabilities of these four highly homologous colicin DNases. Our results indicate that the apo-forms of these colicins are at room temperature and neutral pH in a dynamic conformational equilibrium between at least two quite distinct conformers. We show that the thermal stabilities of the apo-proteins differ by up to 20degreesC. The observed differences correlate with the observed conformational behavior, that is, the tendency of the protein to form either an open, less stable or closed, more stable conformation in solution, as deduced by both tryptophan accessibility studies and electrospray ionization mass spectrometry. Given these surprising structural differences, we next probed the catalytic activity of the four DNases and also observed a significant variation in relative activities. However, no unequivocal link between the activity of the protein and its thermal and structural stability could easily be made. The observed differences in conformational and functional properties of the four colicin DNases are surprising given that they are a closely related ( greater than or equal to65% identity) family of enzymes containing a highly conserved (betabetaalpha-Me) active site motif. The different behavior of the apo-enzymes must therefore most likely depend on more subtle changes in amino acid sequences, most likely in the exosite region (residues 72-98) that is required for specific high-affinity binding of the cognate immunity protein.

Item Type: Article
Copyright, Publisher and Additional Information: Copyright © 2004 The Protein Society.
Institution: The University of York
Academic Units: The University of York > Biology (York)
Depositing User: Christina Hudson
Date Deposited: 20 Feb 2006
Last Modified: 13 Jun 2016 21:43
Published Version: http://dx.doi.org/10.1110/ps.03508204
Status: Published
Refereed: Yes
URI: http://eprints.whiterose.ac.uk/id/eprint/1046

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