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

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.