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Slow conformational dynamics of an endonuclease persist in its complex with its natural protein inhibitor

Published online by Cambridge University Press:  01 April 2000

SARA B.-M. WHITTAKER
Affiliation:
School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
MICHAEL CZISCH
Affiliation:
Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
RAINER WECHSELBERGER
Affiliation:
Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
ROBERT KAPTEIN
Affiliation:
Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
ANDREW M. HEMMINGS
Affiliation:
School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
RICHARD JAMES
Affiliation:
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
COLIN KLEANTHOUS
Affiliation:
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
GEOFFREY R. MOORE
Affiliation:
School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
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Abstract

The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C-terminal DNase domain. Double- and triple-resonance NMR spectra of the isolated DNase domain uniformly labeled with 13C/15N bound to unlabeled Im9 contain more signals than expected for a single DNase conformer, consistent with the bound DNase being present in more than one form. The presence of chemical exchange cross peaks in 750 MHz 15N–1H–15N HSQC–NOESY–HSQC spectra for backbone NH groups of Asp20, Lys21, Trp22, Leu23, Lys69, and Asn70 showed that the bound DNase was in dynamic exchange. The rate of exchange from the major to the minor form was determined to be 1.1 ± 0.2 s−1 at 298 K. Previous NMR studies have shown that the free DNase interchanges between two conformers with a forward rate constant of 1.61 ± 0.11 s−1 at 288 K, and that the bound Im9 is fixed in one conformation. The NMR studies of the bound DNase show that Im9 binds similarly to both conformers of the DNase and that the buried Trp22 is involved in the dynamic process. For the free DNase, all NH groups within a 9 Å radius of any point of the Trp22 ring exhibit heterogeneity suggesting that a rearrangement of the position of this side chain is connected with the conformational interchange. The possible functional significance of this feature of the DNase is discussed.

Type
Research Article
Copyright
© 2000 The Protein Society

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