Residual helical and turn structure in the denatured state of staphylococcal nuclease: analysis of peptide fragments

Abstract

Previous NMR studies of the denatured state of staphylococcal nuclease identified three significantly populated native-like secondary structures: the second alpha-helix, alpha 2 (residues 98-106), estimated to be present 30% of the time, and two highly populated beta-turns, a type I turn (residues 83-86) and a type I' turn (residues 94-97). In the absence of detectable beta-structure or long-range interactions in this low-density denatured state, these three secondary structures appeared to be stabilized exclusively by local interactions. To quantitate the intrinsic stability of these secondary structures, three synthetic peptides corresponding in sequence to these chain segments, plus 2-4 flanking residues, have been analyzed by NMR and CD spectroscopy. Neither of the turn peptides showed significant evidence of residual structure. The data for the alpha 2 peptide suggest that this alpha-helix remains approximately 30% helical when separated from the rest of the protein. However, the type I' turn and the Schellman motif, at the amino and carboxyl termini, respectively, observed in both the native and the denatured state, do not form in this peptide. Instead, the helix appears to propagate to the ends of the peptide, overriding both of these helix-stop signals. The presence of a native-like secondary structure in a denatured protein does not necessarily imply that it has a high intrinsic stability. beta-turns in particular can be stabilized by long-range interactions in the absence of stable beta-strands. In addition, so-called helix-stop signals, such as the Schellman motif, may not contribute actively to helix stability. As for turns, these local interactions at the ends of helices may be passive structures that form in response to longer-range interactions.