The Folding mechanism of barstar: evidence for multiple pathways and multiple intermediates

Shastry, M. C. R. ; Udgaonkar, Jayant B. (1995) The Folding mechanism of barstar: evidence for multiple pathways and multiple intermediates Journal of Molecular Biology, 247 (5). pp. 1013-1027. ISSN 0022-2836

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The mechanism of folding of the small protein barstar in the pre-transition zone at pH 7, 25°C has been characterized using rapid-mixing techniques. Ear;lier studies had established the validity of the three-state US⇌UF⇌N mechanism for folding and unfolding in the presence of guanidine hydrochloride (GdnHCl) at concentrations greater than 2.0 M, where USand UF are the slow-refolding and fast-refolding unfolded forms, respectively, and N is the fully folded form. It is now shown that early intermediates, IS1 and IS2as well as a late native-like intermediate, IN, are present on the folding pathways of US, and an early intermediate IF1on folding pathway of UF, when barstar is refolded in concentrations of GdnHCl below 2.0 M. The rates of formation and disappearance of IN, and the rates of formation of N at three different concentrations of GdnHCl in the pre-transition zone have been measured. The data indicate that in 1.5 M GdnHCl, INis not fully populated on the US→IS1→IN→N pathway because the rate of its formation is so slow that the US⇌ UF⇌N pathway can effectively compete with that pathway. In 1.0 M GdnHCl, the US→IS1→INtransition is so fast that IN is fully populated. In 0.6 M GdnHCl, IN appears not to be fully populated because an alternative folding pathway, US→IS2→N, becomes available for the folding of US, in addition to the US→IS1→IN→N pathway. Measurement of the binding of the hydrophobic dye 1-anilino-8-naphthalenesulphonate (ANS) during folding indicates that ANS binds to two distinct intermediates, IM1and IM2, that form within 2 ms on the US→IM1→IS1→IN→N and US→IM2→IS2→N pathways. There is no evidence for the accumulation of intermediates that can bind ANS on the folding pathway of UF.

Item Type:Article
Source:Copyright of this article belongs to Elsevier Science.
Keywords:Barstar; Folding Kinetics; Protein Folding; Molten Globule; ANS
ID Code:54324
Deposited On:11 Aug 2011 11:12
Last Modified:11 Aug 2011 11:12

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