Improved NMR spectra of a protein-DNA complex through rational mutagenesis and the application of a sensitivity optimized isotope-filtered NOESY experiment

J. Iwahara, J. M. Wojciak, R. T. Clubb

Research output: Contribution to journalArticle

49 Scopus citations

Abstract

The NMR spectra of the complex between the DNA-binding domain of the Dead ringer protein (DRI-DBD, Gly262-Gly398) and its DNA binding site (DRI-DBD:DNA, 26 kDa) have been optimized by biochemical and spectroscopic means. First, we demonstrate the utility of a modified 2D [F1,F2] 13 C-filtered NOESY experiment that employs a 1JHC versus chemical shift optimized adiabatic 13C inversion pulse [Zwahlen, C. et al. (1997) J. Am. Chem. Soc., 119, 6711-6721]. The new sequence is shown to be more sensitive than previously published pulse schemes (up to 40% in favorable cases) and its utility is demonstrated using two protein-DNA complexes. Second, we demonstrate that the targeted replacement of an interfacial aromatic residue in the DRI-DBD:DNA complex substantially reduces line broadening within its NMR spectra. The spectral changes are dramatic, salvaging a protein-DNA complex that was originally ill suited for structural analysis by NMR. This biochemical approach is not a general method, but may prove useful in the spectral optimization of other protein complexes that suffer from interfacial line broadening caused by dynamic changes in proximal aromatic rings.

Original languageEnglish (US)
Pages (from-to)231-241
Number of pages11
JournalJournal of Biomolecular NMR
Volume19
Issue number3
DOIs
StatePublished - Apr 23 2001
Externally publishedYes

Keywords

  • Chemical exchange broadening
  • Isotope-filtered experiment
  • Mutation
  • Protein complex

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy

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