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Related Experiment Videos

Major groove recognition by three-stranded beta-sheets: affinity determinants and conserved structural features.

K M Connolly1, U Ilangovan, J M Wojciak

  • 1Department of Chemistry and Biochemistry and the UCLA-DOE Laboratory of Structural Biology and Genetics, University of California, 405 Hilgard Ave, Los Angeles, CA, 90095-1570, USA.

Journal of Molecular Biology
|July 13, 2000
PubMed
Summary

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Seven protein-DNA hydrogen bonds are crucial for the affinity of the Tn916 integrase DNA-binding domain. These interactions, identified through mutagenesis and binding assays, highlight conserved recognition principles in three-stranded beta-sheet DNA-binding motifs.

Area of Science:

  • Molecular Biology
  • Structural Biology
  • Biochemistry

Background:

  • The Tn916 integrase protein's amino-terminal domain binds DNA via a three-stranded beta-sheet interface.
  • Understanding the specific interactions at this interface is key to deciphering DNA recognition mechanisms.

Purpose of the Study:

  • To investigate the role of interfacial contacts in the binding affinity of the Tn916 integrase-DNA complex.
  • To determine the contribution of specific hydrogen bonds to protein-DNA recognition.

Main Methods:

  • Rational mutagenesis of the integrase protein.
  • Fluorescence anisotropy and intrinsic quenching DNA-binding assays.
  • Analysis of NMR-derived solution structures.

Main Results:

Related Experiment Videos

  • Seven protein-DNA hydrogen bonds, including base-specific and phosphate-interacting ones, significantly contribute to binding affinity.
  • These critical interactions originate from specific strands and loops within the beta-sheet.
  • NMR data indicates that well-defined hydrogen bonds in solution structures are vital for DNA recognition energetics.

Conclusions:

  • The study elucidates the energetic contribution of specific hydrogen bonds to Tn916 integrase-DNA binding.
  • Conserved features in three-stranded beta-sheet DNA-binding motifs suggest common recognition principles across different protein-DNA complexes.