NMR with (13)C, (15)N-doubly-labeled DNA: The shape Antennapedia homeodomain complex with a 14-mer DNA duplex.
Nearly complete (1)H, (13)C and(15) N 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 (15)N-labeled Antennapedia homeodomain. The DNA was either fully (13)C,(15)N-labeled or contained uniformly (13)C, (15)N-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(1) H-(1)H NOEs observed in 3D(13) C-resolved NOESY; and (iii) assignment of the imino and amino groups via (1)H-(1)H NOEs and(15) N-(1)H correlation spectroscopy. The assignment of the duplex in the 17 kDa protein-DNA complex was greatly facilitated by the fact that (1)H signals of the protein were filtered out in (13)C-resolved spectroscopy and by the excellent carbon chemical shift dispersion of the DNA duplex. Comparison of corresponding (13)C chemical shifts of the free and the protein-bound DNA indicates conformational changes in the DNA upon complex formation.
Fernández
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C
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093, Zürich, Switzerland.
Szyperski
T
T
Ono
A
A
Iwai
H
H
Tate
S
S
Kainosho
M
M
Wüthrich
K
K