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

Abstract

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.

Cis proline mutants of ribonuclease A. I. thermal stability

“…This behavior has been observed both in nuclease (Evans et al, 1987(Evans et al, , 1989 and in calbindin (Wendt et al, 1988;Skelton et al, 1990). In these instances, the Gibbs energy difference between the two folded conformations is small and a substituted amino acid is therefore likely to form a trans peptide bond.…”

Stress and strain in staphylococcal nuclease

“…Nuclease A and K116A adopt a cis peptide bond between residues 116 and 117, whereas in short peptides (Grathwohl & Wuthrich, 1976), in peptide analogues of this sequence (Raleigh et al, 1992), and in the unfolded nuclease (Evans et al, 1989) the trans X-Pro peptide bond is favored. One explanation for this phenomenon is that the position of the ends of the loop only allow trans conformations where the local backbone interactions associated with the 9 and rl, torsion angles are strained.…”

The Pro117 to glycine mutation of staphylococcal nuclease simplifies the unfolding—folding kinetics

“…Nevertheless, a question may also be raised on the above explanation of the biphasic kinetics. The isomerization from N, to N, studied by NMR is known to be temperature-dependent with a dH of 4.5 to 54 kJ/mol [4,5]. The temperature dependence has suggested that only N, is populated at 4.YC where the biphasic unfolding was observed by CD.…”