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

J Am Chem Soc. 2004 Oct 13;126(40):12800-8. doi: 10.1021/ja046246b.


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 (1)H paramagnetic relaxation enhancement (PRE) arising from Mn(2+) chelated to an EDTA-group covalently attached to a thymine base (dT-EDTA-Mn(2+)) 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 (1)H-PRE profiles are observed on the (1)H resonances of the bound protein when dT-EDTA-Mn(2+) 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 (1)H-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 (1)H-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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • DNA / chemistry*
  • DNA / metabolism
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism
  • Electron Spin Resonance Spectroscopy
  • Fluorescence Resonance Energy Transfer
  • HMGB1 Protein / chemistry*
  • HMGB1 Protein / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Nuclear Proteins / chemistry*
  • Nuclear Proteins / metabolism
  • Nucleic Acid Conformation
  • Protein Binding
  • Sequence Alignment
  • Sex-Determining Region Y Protein
  • Transcription Factors / chemistry*
  • Transcription Factors / metabolism


  • DNA-Binding Proteins
  • HMGB1 Protein
  • Nuclear Proteins
  • Sex-Determining Region Y Protein
  • Transcription Factors
  • DNA