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We have determined the solution structures and examined the dynamics of the
Escherichia coli trp repressor (a 25-kDa dimer), with and without the
co-repressor L-tryptophan, from NMR data. This is the largest protein structure
thus far determined by NMR. To obtain a set of data sufficient for a structure
determination it was essential to resort to isotopic spectral editing. Line
broadening observed in this molecular mass range precludes for the most part the
measurement of coupling constants and stereospecific assignments, with the
inevitable result that the attainable resolution of the final structure will be
somewhat lower than the resolution reported for smaller proteins and peptides.
Nevertheless the general topology of the protein can be deduced from the subsets
of NOEs defining the secondary and tertiary structure, providing a basis for
further refinement using the full set of NOEs and energy minimization. We report
here (a) an intermediate resolution structure that can be deduced from NMR data,
covalent, angular and van-der-Waals constraints only, without resort to detailed
energy calculations, and (b) the limits of uncertainty within which this
structure is valid. An examination of these structures combined with backbone
amide exchange data shows that even at this resolution three important
conclusions can be drawn: (a) the protein structure changes upon binding
tryptophan; (b) the putative DNA binding region is much more flexible than the
core of the molecule, with backbone amide proton exchange rates 1000 times
faster than in the core; (c) the binding of tryptophan stabilizes the repressor
molecule, which is reflected in both the appearance of additional NOEs, and in
the slowing of backbone proton exchange rates by factors of 3-10.
Sequence-specific 1H-NMR assignments and the secondary structure of the
holopressor (L-tryptophan-bound form) have been reported previously [C. H.
Arrowsmith, R. Pachter, R. B. Altman, S. B. Iyer & O. Jardetzky (1990)
Biochemistry 29, 6332-6341]. Those for the trp aporepressor (L-tryptophan-free
form), made using the same methods and conditions as described in the cited
paper, are reported here. The secondary structure of the aporepressor was
calculated from sequential and medium-range NOEs and is the same as reported for
the holorepressor except that helix E is shorter. The tertiary solution
structures for both forms of the repressor were calculated from long-range NOE
data.(ABSTRACT TRUNCATED AT 400 WORDS)
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