Influence of an extrinsic cross-link on the folding pathway of ribonuclease A. Kinetics of folding-unfolding.
The kinetics of folding/unfolding of cross-linked Lys7-dinitrophenylene-Lys41-ribonuclease A were studied and compared to those of unmodified ribonuclease A (RNase A) at various concentrations of guanidine hydrochloride. The folding of the denatured cross-linked protein involved one fast-folding species (22 +/- 4%) and two slow-folding species, as observed in unmodified ribonuclease A. Also, a nativelike intermediate, analogous to that reported previously for unmodified ribonuclease A [Cook, K. H., Schmid, F. X., & Baldwin, R. L. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6157], has been detected on the folding pathway of cross-linked ribonuclease A. The extrinsic cross-link between Lys7 and Lys41 did not affect the rate constants for the folding kinetics of these three species. The cross-link did, however, significantly affect the rate constant for unfolding of the native protein. The conformation of the protein in the transition state of the unfolding pathway was deduced from an analysis of the kinetic data. It appears that the 41 N-terminal residues are unfolded in the transition state of the unfolding pathway. Thus, the unfolding pathway of RNase A is sequential in that further unfolding (after the transition state) follows the unfolding of the 41 N-terminal residues. Also, the conformation of the 41 N-terminal residues does not play a role in the folding pathway. Presumably, if the cross-link were introduced instead between two other residues that are in the segment(s) involved in the rate-limiting step(s), it could increase the refolding rate constants and possibly the concentration of fast-folding species.
D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004140:Dinitrophenols; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D013816:Thermodynamics
D003432:Cross-Linking Reagents; D004140:Dinitrophenols; D006146:Guanidines; D012259:Ribonuclease, Pancreatic; D019791:Guanidine