A magnetization-transfer nuclear magnetic resonance study of the folding of staphylococcal nuclease.

The equilibrium between alternative folded states of a globular protein, staphylococcal nuclease, has been investigated by using 1H NMR. Magnetization-transfer experiments have revealed the existence of a related structural heterogeneity of the unfolded state, and quantitative analysis of a series of these experiments has permitted the kinetics of folding and interconversion of the different states to be explored. A model based on cis/trans isomerism at the peptide bond preceding Pro-117 has been developed to account for the results. This model, recently supported by a protein-engineering experiment [Evans et al. (1987) Nature (London) 329, 266], has been used to interpret the kinetic data, providing insight into the nature of the folding processes. The predominance of the cis-proline form in the folded state is shown to derive from a large favorable enthalpy term resulting from more effective overall folding interactions. The kinetics of folding and isomerization are shown to occur on similar time scales, such that more than one pathway between two states may be significant. It has been possible, however, to compare the direct folding and unfolding rates within the cis- and trans-proline-containing populations, with results suggesting that the specific stabilization of the cis peptide bond is effective only at a late stage in the folding process.