Characterization of nonspecific protein-DNA interactions by 1H paramagnetic relaxation enhancement

Junji Iwahara, Charles D. Schwieters, G. Marius Clore

Research output: Contribution to journalArticle

61 Scopus citations

Abstract

Nonspecific protein-DNA interactions play an important role in a variety of contexts related to DNA packaging, nucleoprotein complex formation, and gene regulation. Biophysical characterization of nonspecific protein-DNA interactions at the atomic level poses significant challenges owing to the dynamic nature of such complexes. Although NMR spectroscopy represents a powerful tool for the analysis of dynamic systems, conventional NMR techniques have provided little information on nonspecific protein-DNA interactions. We show that intermolecular 1H paramagnetic relaxation enhancement (PRE) arising from Mn 2+ chelated to an EDTA-group covalently attached to a thymine base (dT-EDTA-Mn2+) in DNA provides a unique approach for probing the global dynamics and equilibrium distribution of nonspecific protein-DNA interactions. For nonspecific DNA binding, similar intermolecular 1H-PRE profiles are observed on the 1H resonances of the bound protein when dT-EDTA-Mn2+ is located at either end of a DNA oligonucleotide duplex. We demonstrate the applicability of this approach to HMG-box proteins and contrast the results obtained for nonspecific DNA binding of the A-box of HMGB-1 (HMGB-1A) with sequence-specific DNA binding of the related SRY protein. Intermolecular 1H-PRE data demonstrate unambiguously that HMGB-1A binds to multiple sites in multiple orientations even on a DNA fragment as short as 14 base pairs. Combining the 1H-PRE data with the crystal structure of the HMGB-1 A-box/cisplatin-modified DNA complex allows one to obtain a semiquantitative estimate of the equilibrium populations at the various sites.

Original languageEnglish (US)
Pages (from-to)12800-12808
Number of pages9
JournalJournal of the American Chemical Society
Volume126
Issue number40
StatePublished - Oct 13 2004
Externally publishedYes

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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