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., & Shor tie, D. (1994) Biochemistry 33, 1063-1072], NMR methods were used to c haracterize the residual structure in Delta 131 Delta, a large fragmen t of staphylococcal nuclease that serves as a model denatured state un der nondenaturing conditions. On the basis of a large number of missin g amide protons for the residues that form a three-strand antiparallel beta sheet in the native state, it was concluded that this beta meand er may be highly populated in Delta 131 Delta, with severe line broade ning due to relatively slow exchange between different conformational states. In the present report, results from circular dichroism spectro scopy and NMR spectroscopy indicate strands beta 2-beta 3 form a beta hairpin at urea concentrations below 6 M. Amide proton resonances from several hydrophobic residues adjacent to this beta hairpin disappear in concert with all of the beta 2-beta 3 residues, suggesting a local, non-native hydrophobic interaction may help stabilize the beta hairpi n. At concentrations below 3 M, all amide resonances from strand beta 1 in Delta 131 Delta also disappear, suggesting that beta 1 may combin e with the beta 2-beta 3 hairpin to form a native-like beta meander. I n addition, the hydrophobic helix alpha 2 decreases from approximately 30% population in 0 M urea to approximately 10%-15% at 6 M urea, wher eas helix alpha 1 goes from 10%-15% populated in 0 M urea to undetecta ble in 6 M urea. Characterization of a second, distinctly different de natured state, WT nuclease at pH 3.0 and low salt, reveals that this l ow-density acid-denatured state is structurally similar to Delta 131 D elta at low concentrations of urea. From these and previously publishe d data, a tenative equilibrium folding pathway can be constructed for staphylococcal nuclease which describes the relative strengths and int erdependencies of the chain-chain interactions involved in forming the native state.