NMR with 13C, 15N-doubly-labeled DNA: the Antennapedia homeodomain complex with a 14-mer DNA duplex

J Biomol NMR. 1998 Jul;12(1):25-37.

Abstract

Nearly complete 1H, 13C and 15N NMR assignments have been obtained for a doubly labeled 14-base pair DNA duplex in solution both in the free state and complexed with the uniformly 15N-labeled Antennapedia homeodomain. The DNA was either fully 13C, 15N-labeled or contained uniformly 13C, 15N-labeled nucleotides only at those positions which form the protein-DNA interface in the previously determined NMR solution structure of the Antennapedia homeodomain-DNA complex. The resonance assignments were obtained in three steps: (i) identification of the deoxyribose spin systems via scalar couplings using 2D and 3D HCCH-COSY and soft-relayed HCCH-COSY; (ii) sequential assignment of the nucleotides via 1H-1H NOEs observed in 3D 13C-resolved NOESY; and (iii) assignment of the imino and amino groups via 1H-1H NOEs and 15N-1H correlation spectroscopy. The assignment of the duplex in the 17 kDa protein-DNA complex was greatly facilitated by the fact that 1H signals of the protein were filtered out in 13C-resolved spectroscopy and by the excellent carbon chemical shift dispersion of the DNA duplex. Comparison of corresponding 13C chemical shifts of the free and the protein-bound DNA indicates conformational changes in the DNA upon complex formation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antennapedia Homeodomain Protein
  • Base Sequence
  • Carbon Isotopes
  • DNA / chemistry*
  • DNA / genetics
  • Homeodomain Proteins / genetics*
  • Nitrogen Isotopes
  • Nuclear Magnetic Resonance, Biomolecular / methods
  • Nuclear Proteins*
  • Nucleic Acid Conformation*
  • Nucleic Acid Heteroduplexes / chemistry*
  • Nucleic Acid Heteroduplexes / genetics
  • Nucleoproteins / genetics
  • Oligodeoxyribonucleotides
  • Point Mutation
  • Transcription Factors*

Substances

  • Antennapedia Homeodomain Protein
  • Carbon Isotopes
  • Homeodomain Proteins
  • Nitrogen Isotopes
  • Nuclear Proteins
  • Nucleic Acid Heteroduplexes
  • Nucleoproteins
  • Oligodeoxyribonucleotides
  • Transcription Factors
  • DNA