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Protein Science (2004), 13:1391-1401. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Distinct conformational stability and functional activity of four highly homologous endonuclease colicins

Ewald T.J. van den Bremer1, Anthony H. Keeble2, Wim Jiskoot3, Robin E.J. Spelbrink1, Claudia S. Maier1, Arie Van Hoek4, Antonie J.W.G. Visser4, Richard James5, Geoffrey R. Moore6, Colin Kleanthous2 and Albert J.R. Heck1

1 Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research & Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 3584 CA Utrecht, The Netherlands
2 Department of Biology, University of York, York YO10 5YW, United Kingdom
3 Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 3584 CA Utrecht, The Netherlands
4 MicroSpectroscopy Centre, Laboratories of Biochemistry and Biophysics, Wageningen University, 6703 HA Wageningen, The Netherlands
5 Division of Microbiology & Infectious Diseases, School of Molecular Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH, United Kingdom
6 School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom

(RECEIVED November 5, 2003; FINAL REVISION January 30, 2004; ACCEPTED February 9, 2004)



Abstract

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 20°C. 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 (>=65% identity) family of enzymes containing a highly conserved ({beta}{beta}{alpha}-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.

Keywords: colicins; endonucleases; protein folding; conformational stability; ESI-MS

Reprint requests to: Ewald T.J. van den Bremer, Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research & Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands; e-mail: e.t.j. vandenbremer{at}chem.uu.nl; fax: 31-30-251-8219.

Article and publication are at http://www.proteinsci.org/cgi/doi/10.1110/ps.03508204.


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