Section 69
Chapter 68,519

Recombination of S-peptide with S-protein during folding of ribonuclease S. I. Folding pathways of the slow-folding and fast-folding classes of unfolded S-protein

Labhardt, A.M.; Baldwin, R.L.

Journal of Molecular Biology 135(1): 231-244


ISSN/ISBN: 0022-2836
PMID: 43398
DOI: 10.1016/0022-2836(79)90349-8
Accession: 068518783

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The refolding kinetics of RNase S were measured by tyrosine absorbance, by tyrosine fluorescence emission and by rapid binding of the specific inhibitor 2'CMP to folded [bovine pancreatic] RNase S. The S-protein is 1st unfolded at pH 1.7 and then either mixed with S-peptide as refolding is initiated by a stopped-flow pH jump to pH 6.8, or the same results are obtained if S-protein and S-peptide are present together before refolding is initiated. The refolding kinetics of RNase S were measured as a function of temperature (10-40.degree. C) and of protein concentration (10-120 .mu.M). The results are compared to the folding kinetics of S-protein alone and to earlier studies of RNase A. A thermal folding transition of S-protein was found below 30.degree. C at pH 1.7. The refolding kinetics of unfolded S-protein as it is found above 30.degree. C at pH 1.7, is characterized together with the kinetics of combination between S-peptide and S-protein during folding at pH 6.8. Two classes of unfolded S-protein molecules are found, fast-folding and slow-folding molecules, in a 20:80 ratio. This is the same result as that found earlier for RNase A; it is expected if the slow-folding molecules are produced by the slow cis-trans isomerization of proline residues after unfolding, since S-protein contains all 4 proline residues of RNase A. The refolding kinetics of the fast-folding molecules show clearly that combination between S-peptide and S-protein occurs before folding of S-protein is complete. If combination occurred only after complete folding, then the kinetics of formation of RNase S should be rather slow (5s and 100s at 30.degree. C) and nearly independent of protein concentration, as shown by separate measurements of the folding kinetics of S-protein, and of the combination between S-peptide and folded S-protein. The observed folding kinetics are faster than predicted by this model and also the folding rate increases strongly with protein concentration (apparent 1.6 order kinetics). The fact that RNase S is formed more rapidly than S-protein alone is sufficient by itself to show that combination with S-peptide precedes complete folding of S-protein. Computer simulation of a simple, parallel-pathway scheme reproduces the folding kinetics of the fast-folding molecules. All 3 probes give the same folding kinetics. These results exclude the model for protein folding in which the rate-limiting step is an initial diffusion of the polypeptide chain into a restricted range of 3-dimensional configurations (nucleation) followed by rapid folding (propagation). If this model were valid, comparable rates of folding for RNase A and for S-protein and no populated folding intermediates, so that combination between S-peptide and S-protein should occur after folding is complete. Instead, RNase A folds 60 times more rapidly than S-protein and also combination with S-peptide occurs before folding of S-protein is complete. The folding rate of S-protein increases after the formation, or stabilization, of an intermediate which results from combination with S-peptide. They support a sequential model for protein folding where the rates of successive steps in folding depend on the stabilites of preceding intermediates. The refolding kinetics of the slow-folding molecules are complex. two results demonstrate the presence of folding intermediates: the 3 probes show different kinetic progress curves, and the folding kinetics are concentration-dependent, in contrast to the results expected if complete folding of S-protein precedes combination with S-peptide. A faster phase of the slow-refolding reaction is detected both by tyrosine absorbance and fluorescence emission but not by 2'CMP binding, indicating that native RNase S is not formed in this phase. Comparison of the kinetic progress curves measured by different probes is made with the use of the kinetic ratio test, which is defined here.

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