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Title
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1H NMR studies of the glucocorticoid receptor DNA-binding domain: sequential assignments and identification of secondary structure elements.
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Authors
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T.Härd,
E.Kellenbach,
R.Boelens,
R.Kaptein,
K.Dahlman,
J.Carlstedt-Duke,
L.P.Freedman,
B.A.Maler,
E.I.Hyde,
J.A.Gustafsson.
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Ref.
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Biochemistry, 1990,
29,
9015-9023.
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PubMed id
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Abstract
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Two protein fragments containing the DNA-binding domain (DBD) of the
glucocorticoid receptor (GR) have been studied by two-dimensional 1H NMR
spectroscopy. The two peptides (93 and 115 residues, respectively) contain a
common segment corresponding to residues C440-I519 of the rat GR or residues
C421-I500 of the human GR and include two Zn-binding "finger" domains. The
structures of this segment are almost identical in the two protein fragments, as
judged from chemical shifts and sequential NOE connectivities. More than 90% of
all observable 1H resonances within a 71-residue segment encompassing C440-R510
(rat GR) could be sequentially assigned by standard techniques, and
stereospecific assignments could be made for the methyl groups in four valine
residues within this segment. Sequential NOE connectivities indicate several
elements of secondary structure including two alpha-helical segments consisting
of residues S459-E469 and P493-G504, a type I reverse turn between residues R479
and C482, a type II reverse turn between residues L475 and G478, and several
regions of extended peptide conformation. No evidence for alpha-helical
conformation was found within the two putative zinc-finger domains, indicating
that the structures of these domains differ from that of TFIIIA-type zinc
fingers. The observation of some very slowly exchanging amide protons in the
N-terminal (CI) domain of the DBD in combination with slow rotation of the Y452
aromatic ring indicates that this domain has a restricted conformational
flexibility compared to the C-terminal (CII) domain. We also observe several
long-range NOE connectivities within C440-R510, suggesting that the sequential
assignments presented here will provide a basis for a complete structure
determination of this segment of the GR.
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