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Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates.
J Mol Biol. 2002 Jan 11; 315(2):213-27.JM

Abstract

The Saccharomyces cerevisiae non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro. However, the molecular mechanism of the prion-like activity remains obscure. Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD. The kinetic folding behaviour of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2. Both unfolding and refolding kinetics are multiphasic. An intermediate is populated during unfolding at high denaturant concentrations resulting in the appearance of an unfolding burst phase and "roll-over" in the denaturant dependence of the unfolding rate constants. During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing. A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate. Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate. Double-jump experiments indicate that isomerisation of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence. During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate. In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed.

Authors+Show Affiliations

Centre for Protein Engineering, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.No affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

11779240

Citation

Galani, Despina, et al. "Folding of the Yeast Prion Protein Ure2: Kinetic Evidence for Folding and Unfolding Intermediates." Journal of Molecular Biology, vol. 315, no. 2, 2002, pp. 213-27.
Galani D, Fersht AR, Perrett S. Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates. J Mol Biol. 2002;315(2):213-27.
Galani, D., Fersht, A. R., & Perrett, S. (2002). Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates. Journal of Molecular Biology, 315(2), 213-27.
Galani D, Fersht AR, Perrett S. Folding of the Yeast Prion Protein Ure2: Kinetic Evidence for Folding and Unfolding Intermediates. J Mol Biol. 2002 Jan 11;315(2):213-27. PubMed PMID: 11779240.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates. AU - Galani,Despina, AU - Fersht,Alan R, AU - Perrett,Sarah, PY - 2002/1/10/pubmed PY - 2002/2/8/medline PY - 2002/1/10/entrez SP - 213 EP - 27 JF - Journal of molecular biology JO - J Mol Biol VL - 315 IS - 2 N2 - The Saccharomyces cerevisiae non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro. However, the molecular mechanism of the prion-like activity remains obscure. Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD. The kinetic folding behaviour of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2. Both unfolding and refolding kinetics are multiphasic. An intermediate is populated during unfolding at high denaturant concentrations resulting in the appearance of an unfolding burst phase and "roll-over" in the denaturant dependence of the unfolding rate constants. During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing. A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate. Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate. Double-jump experiments indicate that isomerisation of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence. During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate. In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/11779240/Folding_of_the_yeast_prion_protein_Ure2:_kinetic_evidence_for_folding_and_unfolding_intermediates_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022283602952344 DB - PRIME DP - Unbound Medicine ER -