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.
Fernández
C
C
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland.
Szyperski
T
T
Ono
A
A
Iwai
H
H
Tate
S
S
Kainosho
M
M
Wüthrich
K
K
D051943:Antennapedia Homeodomain Protein; D001483:Base Sequence; D002247:Carbon Isotopes; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry* / Q000235:genetics; D009698:Nucleoproteins / Q000235:genetics; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D014157:Transcription Factors
D051943:Antennapedia Homeodomain Protein; D002247:Carbon Isotopes; D018398:Homeodomain Proteins; D009587:Nitrogen Isotopes; D009687:Nuclear Proteins; D009692:Nucleic Acid Heteroduplexes; D009698:Nucleoproteins; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; D004247:DNA