Cookies on this website
We use cookies to ensure that we give you the best experience on our website. If you click 'Continue' we'll assume that you are happy to receive all cookies and you won't see this message again. Click 'Find out more' for information on how to change your cookie settings.

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 degrees 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 (> or =65% 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.

Original publication

DOI

10.1110/ps.03508204

Type

Journal article

Journal

Protein Sci

Publication Date

05/2004

Volume

13

Pages

1391 - 1401

Keywords

Amino Acid Sequence, Binding Sites, Calorimetry, Colicins, Endodeoxyribonucleases, Enzyme Stability, Fluorescence, Molecular Sequence Data, Sequence Alignment, Spectrometry, Mass, Electrospray Ionization, Structural Homology, Protein, Tryptophan