Amino acid insertion reveals a necessary three-helical intermediate in the folding pathway of the colicin E7 immunity protein Im7.
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.
Knowling
Stuart E
SE
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Figueiredo
Angelo Miguel
AM
Whittaker
Sara B-M
SB
Moore
Geoffrey R
GR
Radford
Sheena E
SE
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003087:Colicins / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry
D000596:Amino Acids; D002352:Carrier Proteins; D003087:Colicins; C509253:E colicin-binding immunity protein Im7, E coli; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins