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Sequence-specific 1H NMR assignments are reported for the active
L-tryptophan-bound form of Escherichia coli trp repressor. The repressor is a
symmetric dimer of 107 residues per monomer; thus at 25 kDa, this is the largest
protein for which such detailed sequence-specific assignments have been made. At
this molecular mass the broad line widths of the NMR resonances preclude the use
of assignment methods based on 1H-1H scalar coupling. Our assignment strategy
centers on two-dimensional nuclear Overhauser spectroscopy (NOESY) of a series
of selectively deuterated repressor analogues. A new methodology was developed
for analysis of the spectra on the basis of the effects of selective deuteration
on cross-peak intensities in the NOESY spectra. A total of 90% of the backbone
amide protons have been assigned, and 70% of the alpha and side-chain proton
resonances are assigned. The local secondary structure was calculated from
sequential and medium-range backbone NOEs with the double-iterated Kalman filter
method [Altman, R. B., & Jardetzky, O. (1989) Methods Enzymol. 177,
218-246]. The secondary structure agrees with that of the crystal structure
[Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L., & Sigler, P.
B. (1985) Nature 317, 782], except that the solution state is somewhat more
disordered in the DNA binding region and in the N-terminal region of the first
alpha-helix. Since the repressor is a symmetric dimer, long-range intersubunit
NOEs were distinguished from intrasubunit interactions by formation of
heterodimers between two appropriate selectively deuterated proteins and
comparison of the resulting NOESY spectrum with that of each selectively
deuterated homodimer. Thus, from spectra of three heterodimers, long-range NOEs
between eight pairs of residues were identified as intersubunit NOEs, and two
additional long-range intrasubunits NOEs were assigned.
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