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

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

Research output: Contribution to journalArticle

62 Citations (Scopus)

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

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HMGB Proteins
DNA
Proteins
Ethylenediaminetetraacetic acid
DNA Packaging
Nucleoproteins
Thymine
Systems Analysis
Edetic Acid
Oligonucleotides
Base Pairing
Cisplatin
Gene expression
Magnetic Resonance Spectroscopy
Nuclear magnetic resonance spectroscopy
Packaging
Dynamical systems
Crystal structure
Nuclear magnetic resonance
Population

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Characterization of nonspecific protein-DNA interactions by 1H paramagnetic relaxation enhancement. / Iwahara, Junji; Schwieters, Charles D.; Clore, G. Marius.

In: Journal of the American Chemical Society, Vol. 126, No. 40, 13.10.2004, p. 12800-12808.

Research output: Contribution to journalArticle

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