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Solution structure and stability of the full‐length excisionase from bacteriophage HK022
Author(s) -
Rogov Vladimir V.,
Lücke Christian,
Muresanu Lucia,
Wienk Hans,
Kleinhaus Ioana,
Werner Karla,
Löhr Frank,
Pristovšek Primož,
Rüterjans Heinz
Publication year - 2003
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.2003.03884.x
Subject(s) - crystallography , chemistry , protein secondary structure , heteronuclear molecule , asparagine , nuclear magnetic resonance spectroscopy , bacteriophage , heteronuclear single quantum coherence spectroscopy , denaturation (fissile materials) , protein structure , amino acid , stereochemistry , biochemistry , escherichia coli , nuclear chemistry , gene
Heteronuclear high‐resolution NMR spectroscopy was employed to determine the solution structure of the excisionase protein (Xis) from the λ‐like bacteriophage HK022 and to study its sequence‐specific DNA interaction. As wild‐type Xis was previously characterized as a generally unstable protein, a biologically active HK022 Xis mutant with a single amino acid substitution Cys28→Ser was used in this work. This substitution has been shown to diminish the irreversibility of Xis denaturation and subsequent degradation, but does not affect the structural or thermodynamic properties of the protein, as evidenced by NMR and differential scanning calorimetry. The solution structure of HK022 Xis forms a compact, highly ordered protein core with two well‐defined α‐helices (residues 5–11 and 18–27) and five β‐strands (residues 2–4, 30–31, 35–36, 41–44 and 48–49). These data correlate well with 1 H 2 O‐ 2 H 2 O exchange experiments and imply a different organization of the HK022 Xis secondary structure elements in comparison with the previously determined structure of the bacteriophage λ excisionase. Superposition of both Xis structures indicates a better correspondence of the full‐length HK022 Xis to the typical ‘winged‐helix’ DNA‐binding motif, as found, for example, in the DNA‐binding domain of the Mu‐phage repressor. Residues 51–72, which were not resolved in the λ Xis, do not show any regular structure in HK022 Xis and thus appear to be completely disordered in solution. The resonance assignments have shown, however, that an unusual connectivity exists between residues Asn66 and Gly67 owing to asparagine‐isoaspartyl isomerization. Such an isomerization has been previously observed and characterized only in eukaryotic proteins.

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