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Title
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Backbone dynamics of the glucocorticoid receptor DNA-binding domain.
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Authors
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H.Berglund,
H.Kovács,
K.Dahlman-Wright,
J.A.Gustafsson,
T.Härd.
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Ref.
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Biochemistry, 1992,
31,
12001-12011.
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PubMed id
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Abstract
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The extent of rapid (picosecond) backbone motions within the glucocorticoid
receptor DNA-binding domain (GR DBD) has been investigated using proton-detected
heteronuclear NMR spectroscopy on uniformly 15N-labeled protein fragments
containing the GR DBD. Sequence-specific 15N resonance assignments, based on
two- and three-dimensional heteronuclear NMR spectra, are reported for 65 of 69
backbone amides within the segment C440-A509 of the rat GR in a protein fragment
containing a total of 82 residues (MW = 9200). Individual backbone 15N
spin-lattice relaxation times (T1), rotating-frame spin-lattice relaxation times
(T1 rho), and steady-state (1H)-15N nuclear Overhauser effects (NOEs) have been
measured at 11.74 T for a majority of the backbone amide nitrogens within the
segment C440-N506. T1 relaxation times and NOEs are interpreted in terms of a
generalized order parameter (S2) and an effective correlation time (tau e)
characterizing internal motions in each backbone amide using an optimized value
for the correlation time for isotropic rotational motions of the protein (tau R
= 6.3 ns). Average S2 order parameters are found to be similar (approximately
0.86 +/- 0.07) for various functional domains of the DBD. Qualitative inspection
as well as quantitative analysis of the relaxation and NOE data suggests that
the picosecond flexibility of the DBD backbone is limited and uniform over the
entire protein, with the possible exception of residues S448-H451 of the first
zinc domain and a few residues for which relaxation and NOE parameters were not
obtained. in particular, we find no evidence for extensive rapid backbone
motions within the second zinc domain. Our results therefore suggest that the
second zinc domain is not disordered in the uncomplexed state of DBD, although
the possibility of slowly exchanging (ordered) conformational states cannot be
excluded in the present analysis.
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