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Distinct conformational stability and functional activity of four highly homologous endonuclease colicins
Author(s) -
van den Bremer Ewald T.J.,
Keeble Anthony H.,
Jiskoot Wim,
Spelbrink Robin E.J.,
Maier Claudia S.,
Van Hoek Arie,
Visser Antonie J.W.G.,
James Richard,
Moore Geoffrey R.,
Kleanthous Colin,
Heck Albert J.R.
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03508204
Subject(s) - colicin , chemistry , conformational isomerism , endonuclease , protein superfamily , protein structure , tryptophan , biophysics , enzyme , biochemistry , crystallography , amino acid , stereochemistry , escherichia coli , biology , molecule , gene , organic chemistry
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 (ββα‐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.