| Structural analyses of denatured proteins have demonstrated that the polypeptide chain does not unfold to a random coil of featureless interactions. Previous work on a highly dynamic denatured fragment of staphylococcal nuclease, termed Δ131Δ, has demonstrated the persistence of native-like, global topology in the absence of fixed hydrogen bonds and tight packing of side-chains. To probe the physical chemistry of this residual structure, long-range structural analyses were carried out on Δ131Δ by measuring residual dipolar couplings as a function of mutations and solution conditions. Couplings were measured for Δ131Δ in H2O, 32°C, pH 5.3 and found to persist at high concentrations of urea, conditions that lead to both increased dynamic motions and a significant expansion of the volume occupied by the polypeptide chain. Comparisons of residual dipolar couplings of full-length, wild-type nuclease in two different denatured states, 4M urea and acidic pH, were made to the fragment in H2O at pH 5.3. To assess the role of hydrophobic interactions, several large nonpolar residues were substituted with polar residues of similar size. Also, the effect of the removal of 30 residues from the N- or C-terminus of Δ131Δ creating two 101-residue fragments was studied. In all cases, the residual dipolar couplings measured for the mutants correlate highly to the couplings measured for Δ131Δ in H2O, pH 5.3, and 32°C. The long-range structure of the denatured state is independent of the mechanism of denaturation and insensitive to any type of long-range interaction that otherwise would have a dominant role in protein stability. We suggest that the native-like topology of the denatured state is primarily encoded by local interactions between side-chains and adjacent backbone segments. |
