Strain in the folding nucleus of chymotrypsin inhibitor 2.
BACKGROUND
Chymotrypsin inhibitor 2 (CI2) is a member of the class of fast-folding small proteins, which is very suitable for testing theories of folding. CI2 folds around a diffuse extended nucleus consisting of the single alpha helix and a set of hydrophobic residues. In particular, Ala16 has been predicted and independently found to interact with Leu49 and Ile57, hydrophobic residues that are highly conserved among homologues. We have characterised in detail the interactions between these residues in the folding nucleus of the protein by using double-mutant cycles.


RESULTS
Surprisingly, we find that there is some destabilising strain in the transition state for folding of the wild-type protein between Ala16 and Ile57. Further, we find that the strain is larger in the native state of the protein. This is shown directly in the unfolding kinetics, which clearly show a release of strain. The net result of this is that the presence of both residues speeds up folding. Ala16 and Leu49 interact favourably in the transition state, but have no net interaction energy in the native state.


CONCLUSIONS
Part of the folding nucleus of the protein fits together more snugly in the transition state than it does in the native state. Interactions between some of the closely packed residues in the folding nucleus of CI2 may perhaps be optimised for the rate of folding and not for stability.
Ladurner
A G
AG
MRC Cambridge Centre for Protein Engineering, MRC Centre, UK.
Itzhaki
L S
LS
Fersht
A R
AR
D000409:Alanine / Q000235:genetics; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D005998:Glycine / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D010940:Plant Proteins; D017510:Protein Folding; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
D010455:Peptides; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; C093336:chymotrypsin inhibitor 2; D002918:Chymotrypsin; D000409:Alanine; D005998:Glycine