Amino Acid Insertion Reveals a Necessary Three-Helical Intermediate in the Folding Pathway of the Colicin E7 Immunity Protein Im7

JOURNAL OF MOLECULAR BIOLOGY

卷:392

Article

Knowling, Stuart E.^1,2  Figueiredo, Angelo Miguel³  Whittaker, Sara B-M.^3,4  Moore, Geoffrey R.³  Radford, Sheena E.^1,2 

[1] Univ Leeds, Astbury Ctr Struct Mol Biol, Leeds LS2 9JT, W Yorkshire, England

[2] Univ Leeds, Inst Mol & Cellular Biol, Leeds LS2 9JT, W Yorkshire, England

[3] Univ E Anglia, Ctr Mol & Struct Biochem, Sch Chem Sci & Pharm, Norwich NR4 7TJ, Norfolk, England

[4] Univ Birmingham, Sch Canc Sci, Birmingham B15 2TT, W Midlands, England

关键词: polyalanine;    protein folding;    phi-value analysis;    helix design;    intermediate;   

DOI  :  10.1016/j.jmb.2009.07.085

来源: Elsevier

【 摘 要 】

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 phi-value analysis, respectively. The results reveal a robust native structure that is not perturbed by the presence of the extended helix M. 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 In. (C) 2009 Elsevier Ltd. All rights reserved.

【 授权许可】

Free