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Kinetic and thermodynamic studies of the folding/unfolding of a tryptophan-containing mutant of ribonuclease A.
Biochemistry. 1996 Oct 01; 35(39):12978-92.B

Abstract

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.

Authors+Show Affiliations

Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8841145

Citation

Sendak, R A., et al. "Kinetic and Thermodynamic Studies of the Folding/unfolding of a Tryptophan-containing Mutant of Ribonuclease A." Biochemistry, vol. 35, no. 39, 1996, pp. 12978-92.
Sendak RA, Rothwarf DM, Wedemeyer WJ, et al. Kinetic and thermodynamic studies of the folding/unfolding of a tryptophan-containing mutant of ribonuclease A. Biochemistry. 1996;35(39):12978-92.
Sendak, R. A., Rothwarf, D. M., Wedemeyer, W. J., Houry, W. A., & Scheraga, H. A. (1996). Kinetic and thermodynamic studies of the folding/unfolding of a tryptophan-containing mutant of ribonuclease A. Biochemistry, 35(39), 12978-92.
Sendak RA, et al. Kinetic and Thermodynamic Studies of the Folding/unfolding of a Tryptophan-containing Mutant of Ribonuclease A. Biochemistry. 1996 Oct 1;35(39):12978-92. PubMed PMID: 8841145.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Kinetic and thermodynamic studies of the folding/unfolding of a tryptophan-containing mutant of ribonuclease A. AU - Sendak,R A, AU - Rothwarf,D M, AU - Wedemeyer,W J, AU - Houry,W A, AU - Scheraga,H A, PY - 1996/10/1/pubmed PY - 1996/10/1/medline PY - 1996/10/1/entrez SP - 12978 EP - 92 JF - Biochemistry JO - Biochemistry VL - 35 IS - 39 N2 - 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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8841145/Kinetic_and_thermodynamic_studies_of_the_folding/unfolding_of_a_tryptophan_containing_mutant_of_ribonuclease_A_ DB - PRIME DP - Unbound Medicine ER -