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Title
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Nuclear magnetic resonance spectroscopy of a DNA complex with the uniformly 13C-labeled Antennapedia homeodomain and structure determination of the DNA-bound homeodomain.
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Authors
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Y.Q.Qian,
G.Otting,
M.Billeter,
M.Müller,
W.Gehring,
K.Wüthrich.
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Ref.
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J Mol Biol, 1993,
234,
1070-1083.
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PubMed id
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Abstract
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A 1:1 complex formed by the mutant Antennapedia(C39S) homeodomain and a 14
base-pair DNA duplex (molecular weight approximately 18,000) was prepared in
which the protein was uniformly 13C-labeled. Using two-dimensional nuclear
Overhauser enhancement (NOE) spectroscopy with 13C(omega 1, omega
2)-double-half-filter and three-dimensional 13C-correlated NOE spectroscopy,
nearly complete sequence-specific resonance assignments were obtained for both
the protein and the DNA in the complex. On this basis conformational constraints
needed for a three-dimensional structure determination were collected. Using 855
intramolecular distance constraints as input, the structure of the DNA-bound
Antp(C39S) homeodomain was calculated with the program DIANA, followed by
restrained energy minimization with the program OPAL. A group of 20 conformers
characterizes a well-defined structure for residues 8 to 56, with an average of
0.5 A of the pairwise root-mean-square deviations calculated for the backbone
atoms of the individual conformers relative to the mean coordinates. The quality
of the resulting structure is comparable to the one for the free protein, and
the global fold of the free Antp(C39S) homeodomain was found to be conserved in
the DNA complex. The structure of the DNA-bound protein was the starting point
for the subsequent structure determination of the complete Antp(C39S)
homeodomain-DNA complex in solution.
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