Amino acid insertion reveals a necessary three-helical intermediate in the folding pathway of the colicin E7 immunity protein Im7
In: Journal of molecular biology, Jg. 392 (2009-06-04), Heft 4
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Zugriff:
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.
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Amino acid insertion reveals a necessary three-helical intermediate in the folding pathway of the colicin E7 immunity protein Im7
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Autor/in / Beteiligte Person: | Knowling, Stuart ; Sara B.-M. Whittaker ; Moore, Geoffrey R. ; Radford, Sheena E. ; Angelo Miguel Figueiredo |
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Zeitschrift: | Journal of molecular biology, Jg. 392 (2009-06-04), Heft 4 |
Veröffentlichung: | 2009 |
Medientyp: | unknown |
ISSN: | 1089-8638 (print) |
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