Kinetic and thermodynamic studies of the folding/unfolding of a tryptophan-containing mutant of ribonuclease A.
Tryptophan was substituted for Tyr92 to create a sensitive and unique optical probe in order to study the unfolding and refolding kinetics of disulfide-intact bovine pancreatic ribonuclease A by fluorescence-detected stopped-flow techniques. The stability of the Trp mutant was found to be similar to that of wild-type RNase A when denatured by heat or GdnHCl, and the mutant was found to have 85% of the activity of the wild-type protein. Single-jump unfolding experiments showed that the unfolding pathway of the Trp mutant contains a fast and a slow phase similar to those seen previously for the wild-type protein, indicating that the mutation did not alter the unfolding pathway significantly. The activation energy of the slow-unfolding phase suggested that proline isomerization is involved, with the Trp residue presumably reporting on changes in its local environment. Single-jump refolding experiments revealed the presence of GdnHCl-independent burst phase and a native-like intermediate, most likely IN, on the folding pathway. Single-jump refolding data at various final GdnHCl concentrations were fit to a kinetic folding model involving two pathways to the native state; one pathway involves the intermediate IN, and the other is a direct one to the native state. This model provides site-specific information, since Trp92 monitors the formation of local structure only in the neighborhood of that residue. Double-jump refolding experiments permitted the detection of a previously reported, hydrophobically collapsed intermediate, I phi. The refolding data support the hypothesis that the region around position 92 is a chain-folding initiation site in the folding pathway.
Sendak
R A
RA
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Rothwarf
D M
DM
Wedemeyer
W J
WJ
Houry
W A
WA
Scheraga
H A
HA
D000818:Animals; D002417:Cattle; D004795:Enzyme Stability; D005453:Fluorescence; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D006146:Guanidines; D014364:Tryptophan; D012259:Ribonuclease, Pancreatic; D019791:Guanidine