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Kinetic studies of the folding of heterodimeric monellin: evidence for switching between alternative parallel pathways.
J Mol Biol. 2012 Jul 13; 420(3):235-50.JM

Abstract

Determining whether or not a protein uses multiple pathways to fold is an important goal in protein folding studies. When multiple pathways are present, defined by transition states that differ in their compactness and structure but not significantly in energy, they may manifest themselves by causing the dependence on denaturant concentration of the logarithm of the observed rate constant of folding to have an upward curvature. In this study, the folding mechanism of heterodimeric monellin [double-chain monellin (dcMN)] has been studied over a range of protein and guanidine hydrochloride (GdnHCl) concentrations, using the intrinsic tryptophan fluorescence of the protein as the probe for the folding reaction. Refolding is shown to occur in multiple kinetic phases. In the first stage of refolding, which is silent to any change in intrinsic fluorescence, the two chains of monellin bind to one another to form an encounter complex. Interrupted folding experiments show that the initial encounter complex folds to native dcMN via two folding routes. A productive folding intermediate population is identified on one route but not on both of these routes. Two intermediate subpopulations appear to form in a fast kinetic phase, and native dcMN forms in a slow kinetic phase. The chevron arms for both the fast and slow phases of refolding are shown to have upward curvatures, suggesting that at least two pathways each defined by a different intermediate are operational during these kinetic phases of structure formation. Refolding switches from one pathway to the other as the GdnHCl concentration is increased.

Authors+Show Affiliations

National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India.No affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

22542529

Citation

Aghera, Nilesh, and Jayant B. Udgaonkar. "Kinetic Studies of the Folding of Heterodimeric Monellin: Evidence for Switching Between Alternative Parallel Pathways." Journal of Molecular Biology, vol. 420, no. 3, 2012, pp. 235-50.
Aghera N, Udgaonkar JB. Kinetic studies of the folding of heterodimeric monellin: evidence for switching between alternative parallel pathways. J Mol Biol. 2012;420(3):235-50.
Aghera, N., & Udgaonkar, J. B. (2012). Kinetic studies of the folding of heterodimeric monellin: evidence for switching between alternative parallel pathways. Journal of Molecular Biology, 420(3), 235-50. https://doi.org/10.1016/j.jmb.2012.04.019
Aghera N, Udgaonkar JB. Kinetic Studies of the Folding of Heterodimeric Monellin: Evidence for Switching Between Alternative Parallel Pathways. J Mol Biol. 2012 Jul 13;420(3):235-50. PubMed PMID: 22542529.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Kinetic studies of the folding of heterodimeric monellin: evidence for switching between alternative parallel pathways. AU - Aghera,Nilesh, AU - Udgaonkar,Jayant B, Y1 - 2012/04/24/ PY - 2011/09/23/received PY - 2012/04/14/revised PY - 2012/04/18/accepted PY - 2012/5/1/entrez PY - 2012/5/1/pubmed PY - 2012/8/21/medline SP - 235 EP - 50 JF - Journal of molecular biology JO - J Mol Biol VL - 420 IS - 3 N2 - Determining whether or not a protein uses multiple pathways to fold is an important goal in protein folding studies. When multiple pathways are present, defined by transition states that differ in their compactness and structure but not significantly in energy, they may manifest themselves by causing the dependence on denaturant concentration of the logarithm of the observed rate constant of folding to have an upward curvature. In this study, the folding mechanism of heterodimeric monellin [double-chain monellin (dcMN)] has been studied over a range of protein and guanidine hydrochloride (GdnHCl) concentrations, using the intrinsic tryptophan fluorescence of the protein as the probe for the folding reaction. Refolding is shown to occur in multiple kinetic phases. In the first stage of refolding, which is silent to any change in intrinsic fluorescence, the two chains of monellin bind to one another to form an encounter complex. Interrupted folding experiments show that the initial encounter complex folds to native dcMN via two folding routes. A productive folding intermediate population is identified on one route but not on both of these routes. Two intermediate subpopulations appear to form in a fast kinetic phase, and native dcMN forms in a slow kinetic phase. The chevron arms for both the fast and slow phases of refolding are shown to have upward curvatures, suggesting that at least two pathways each defined by a different intermediate are operational during these kinetic phases of structure formation. Refolding switches from one pathway to the other as the GdnHCl concentration is increased. SN - 1089-8638 UR - https://www.unboundmedicine.com/medline/citation/22542529/Kinetic_studies_of_the_folding_of_heterodimeric_monellin:_evidence_for_switching_between_alternative_parallel_pathways_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(12)00348-8 DB - PRIME DP - Unbound Medicine ER -