Characterization of nonspecific protein-DNA interactions by 1H paramagnetic relaxation enhancement.
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.
Contribution from the Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, MD 20892-0520, USA.
Junji
J
Iwahara
Charles D
CD
Schwieters
G Marius
GM
Clore
D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D031541:Fluorescence Resonance Energy Transfer; D024243:HMGB1 Protein / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D016415:Sequence Alignment; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; D009687:Nuclear Proteins; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors; D004247:DNA