NMR WITH C-13, N-15-DOUBLY-LABELED DNA - THE ANTENNAPEDIA HOMEODOMAINCOMPLEX WITH A 14-MER DNA DUPLEX

Nearly complete H-1, C-13 and N-15 NMR assignments have been obtained for a doubly labeled 14-base pair DNA duplex in solution both in the f ree state and complexed with the uniformly N-15-labeled Antennapedia h omeodomain. The DNA was either fully C-13, N-15-labeled or contained u niformly C-13, N-15-labeled nucleotides only at those positions which form the protein-DNA interface in the previously determined NMR soluti on structure of the Antennapedia homeodomain-DNA complex. The resonanc e assignments were obtained in three steps: (i) identification of the deoxyribose spin systems via scalar couplings using 2D and 3D HCCH-COS Y and soft-relayed HCCH-COSY; (ii) sequential assignment of the nucleo tides via H-1-H-1 NOEs observed in 3D C-13-resolved NOESY; and (iii) a ssignment of the imino and amino groups via H-1-H-1 NOEs and N-15-H-1 correlation spectroscopy. The assignment of the duplex in the 17 kDa p rotein-DNA complex was greatly facilitated by the fact that H-1 signal s of the protein were filtered out in C-13-resolved spectroscopy and b y the excellent carbon chemical shift dispersion of the DNA duplex. Co mparison of corresponding C-13 chemical shifts of the free and the pro tein-bound DNA indicates conformational changes in the DNA upon comple x formation.