The Equilibrium Folding Pathway of Staphylococcal Nuclease: Identification of the Most Stable Chain-Chain Interactions by NMR and CD Spectroscopy

In a previous report [Alexandrescu, A. T., Abeygunawardana, C., & Shortle, D. (1994) Biochemistry 33, 1063-1072], NMR methods were used to characterize the residual structure in ∆131∆, a large fragment of staphylococcal nuclease that serves as a model denatured state under nondenaturing conditions. On the basis of a large number of missing amide protons for the residues that form a threestrand antiparallel β sheet in the native state, it was concluded that this β meander may be highly populated in A131A, with severe line broadening due to relatively slow exchange between different conformational states. In the present report, results from circular dichroism spectroscopy and NMR spectroscopy indicate strands β2-β3 form a β hairpin at urea concentrations below 6 M. Amide proton resonances from several hydrophobic residues adjacent to this β hairpin disappear in concert with all of the β2-β3 residues, suggesting a local, non-native hydrophobic interaction may help stabilize the beta hairpin. At concentrations below 3 M, all amide resonances from strand β1 in ∆131∆ also disappear, suggesting that β1 may combine with the f32-β3 hairpin to form a native-like β meander. In addition, the hydrophobic helix a.2 decreases from approximately 30% population in 0 M urea to approximately 10%-15% at 6 M urea, whereas helix al goes from 10%-15% populated in 0 M urea to undetectable in 6 M urea. Characterization of a second, distinctly different denatured state, WT nuclease at pH 3.0 and low salt, reveals that this lowdensity acid-denatured state is structurally similar to ∆131∆ at low concentrations of urea. From these and previously published data, a tenative equilibrium folding pathway can be constructed for staphylococcal nuclease which describes the relative strengths and interdependencies of the chain-chain interactions involved in forming the native state.