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Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions

Pashley, CL, Morgan, GJ, Kalverda, AP, Thompson, GS, Kleanthous, C, Radford, SE (2012) Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions. Journal of Molecular Biology, 416 . pp. 300-318. ISSN 0022-2836. (doi:10.1016/j.jmb.2011.12.041) (KAR id:71808)


The unfolded ensemble in aqueous solution represents the starting point of protein folding. Characterisation of this species is often difficult since the native state is usually predominantly populated at equilibrium. Previous work has shown that the four-helix protein, Im7 (immunity protein 7), folds via an on-pathway intermediate. While the transition states and folding intermediate have been characterised in atomistic detail, knowledge of the unfolded ensemble under the same ambient conditions remained sparse. Here, we introduce destabilising amino acid substitutions into the sequence of Im7, such that the unfolded state becomes predominantly populated at equilibrium in the absence of denaturant. Using far- and near-UV CD, fluorescence, urea titration and heteronuclear NMR experiments, we show that three amino acid substitutions (L18A-L19A-L37A) are sufficient to prevent Im7 folding, such that the unfolded state is predominantly populated at equilibrium. Using measurement of chemical shifts, (15)N transverse relaxation rates and sedimentation coefficients, we show that the unfolded species of L18A-L19A-L37A deviates significantly from random-coil behaviour. Specifically, we demonstrate that this unfolded species is compact (R(h)=25 Å) relative to the urea-denatured state (R(h)?30 Å) and contains local clusters of hydrophobic residues in regions that correspond to the four helices in the native state. Despite these interactions, there is no evidence for long-range stabilising tertiary interactions or persistent helical structure. The results reveal an unfolded ensemble that is conformationally restricted in regions of the polypeptide chain that ultimately form helices I, II and IV in the native state.

Item Type: Article
DOI/Identification number: 10.1016/j.jmb.2011.12.041
Subjects: Q Science > QP Physiology (Living systems) > QP517 Biochemistry
Divisions: Divisions > Division of Natural Sciences > Biosciences
Depositing User: Gary Thompson
Date Deposited: 23 Jan 2019 20:49 UTC
Last Modified: 16 Nov 2021 10:26 UTC
Resource URI: (The current URI for this page, for reference purposes)

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