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Slow conformational dynamics of an endonuclease persist in its complex with its natural protein inhibitor
Author(s) -
Whittaker Sara B.M.,
Moore Geoffrey R.,
Hemmings Andrew M.,
Czisch Michael,
Wechselberger Rainer,
Kaptein Robert,
James Richard,
Kleanthous Colin
Publication year - 2000
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.9.4.713
Subject(s) - colicin , conformational isomerism , heteronuclear single quantum coherence spectroscopy , chemistry , stereochemistry , deoxyribonuclease i , crystallography , nuclear magnetic resonance spectroscopy , dna , biochemistry , molecule , organic chemistry , plasmid , base sequence
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 13 C/ 15 N 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 15 N‐ 1 H‐ 15 N 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.