Amino acid insertion reveals a necessary three-helical intermediate in the folding pathway of the colicin E7 immunity protein Im7

Knowling, Stuart E., Figueiredo, Angelo M., Whittaker, Sara B.-M., Moore, Geoffrey R. and Radford, Sheena E. (2009) Amino acid insertion reveals a necessary three-helical intermediate in the folding pathway of the colicin E7 immunity protein Im7. Journal of Molecular Biology, 392 (4). pp. 1074-1086. ISSN 0022-2836

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Abstract

The small (87-residue) alpha-helical protein Im7 (an inhibitor protein for colicin E7 that provides immunity to cells producing colicin E7) folds via a three-state mechanism involving an on-pathway intermediate. This kinetic intermediate contains three of four native helices that are oriented in a non-native manner so as to minimise exposed hydrophobic surface area at this point in folding. The short (6-residue) helix III has been shown to be unstructured in the intermediate ensemble and does not dock onto the developing hydrophobic core until after the rate-limiting transition state has been traversed. After helix III has docked, it adopts an alpha-helical secondary structure, and the side chains of residues within this region provide contacts that are crucial to native-state stability. In order to probe further the role of helix III in the folding mechanism of Im7, we created a variant that contains an eight-amino-acid polyalanine-like helix stabilised by a Glu-Arg salt bridge and an Asn-Pro-Gly capping motif, juxtaposed C-terminal to the natural 6-residue helix III. The effect of this insertion on the structure of the native protein and its folding mechanism were studied using NMR and varphi-value analysis, respectively. The results reveal a robust native structure that is not perturbed by the presence of the extended helix III. Mutational analysis performed to probe the folding mechanism of the redesigned protein revealed a conserved mechanism involving the canonical three-helical intermediate. The results suggest that folding via a three-helical species stabilised by both native and non-native interactions is an essential feature of Im7 folding, independent of the helical propensity of helix III.

Item Type:	Article
Faculty \ School:	Faculty of Science > School of Chemistry
UEA Research Groups:	Faculty of Science > Research Groups > Biophysical Chemistry (former - to 2017)
Depositing User:	Users 2731 not found.
Date Deposited:	14 Mar 2012 15:08
Last Modified:	23 Oct 2022 01:47
URI:	https://ueaeprints.uea.ac.uk/id/eprint/38283
DOI:	10.1016/j.jmb.2009.07.085

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