1H, 15N and 13C NMR assignments of the 434 repressor fragments 1–63 and 44–63 unfolded in 7 M urea

Abstract

An E. coli overexpression system for the N-terminal domain of the 434 repressor with residues 1–63 (434 repressor(1–63)) was constructed and used to produce this polypeptide with uniform 15N-labeling, and with 13C-labeling of the methyl groups of valine and leucine. Using these protein preparations almost complete sequence-specific resonance assignments were obtained for the urea-unfolded form of the 434 repressor(1–63). In addition, the isotope-labeled tryptic peptide, 44–63, was produced by enzymatic cleavage of the recombinant 434 repressor(1–63), and its NMR spectrum was assigned. Corresponding residues in 434 repressor(1–63) and 434 repressor(44–63) in 7 M urea were found to have nearly identical chemical shifts, and in both species similar deviations from 1H random coil shifts were found as previously in 434 repressor(1–69). These indicate the presence of residual non-random structure in the polypeptide segment 50–60. The present NMR assignments, which include stereospecific assignments for the diastereotopic methyl groups of Val and Leu, are the basis for detailed studies of this residual structure in the urea-unfolded form of the 434 repressor.