Initial hydrophobic collapse is not necessary for folding RNase A.

BACKGROUND: One of the main distinctions between different theories describing protein folding is the predicted sequence of secondary structure formation and compaction during the folding process. Whether secondary structure formation precedes compaction of the protein molecules or secondary structure formation is driven by a hydrophobic collapse cannot be decided unequivocally on the basis of existing experimental data. RESULTS: In this study, we investigate the refolding of chemically denatured, disulfide-intact ribonuclease A (RNase A) by monitoring compaction and secondary structure formation using stopped-flow dynamic light scattering and stopped-flow CD, respectively. Our data reveal the formation of a considerable amount of secondary structure early in the refolding of the slow folding species of RNase A without a significant compaction of the molecules. A simultaneous formation of secondary structure and compaction is observed in the subsequent rate-limiting step of folding. CONCLUSIONS: During folding of RNase A an initial global hydrophobicity is not observed, which contradicts the view that this is a general requirement for protein folding. This folding behavior could be typical of similar, moderately hydrophobic proteins.

Reduced-denatured ribonuclease A is not in a compact state.

Dynamic light scattering and circular dichroism experiments were performed to determine the compactness and residual secondary structure of reduced and by 6 M guanidine hydrochloride denatured ribonuclease A. We find that reduction of the four disulphide bonds by dithiothreitol at 20 degrees C leads to total unfolding and that a temperature increase has no further effect on the dimension. The Stokes' radius of ribonuclease A at 20 degrees C is R(s) = (1.90 +/- 0.04) nm (native) and R(s) = (3.14 +/- 0.06) nm (reduced-denatured). Furthermore, circular dichroism spectra do not indicate any residual secondary structure. We suggest that reduced-denatured Ribonuclease A has a random coil-like conformation and is not in a compact denatured state.