Protein fragments as models for events in protein folding pathways: protein engineering analysis of the association of two complementary fragments of the barley chymotrypsin inhibitor 2 (CI-2).
Two fragments of chymotrypsin inhibitor-2, CI-2(20-59) and CI-2(60-83), derived from cyanogen bromide cleavage at Met-59, associate to give a native-like structure. We analyze the kinetics and equilibria of association of mutant fragments derived from cleaving mutant proteins at the same methionine residue. The changes in free energy of association have been measured both from isothermal studies of the binding of fragments and from thermal denaturation of the complexes. In general, there is a good correlation between the changes on mutation of the free energy of association of fragments and the changes in free energy of folding of the uncleaved parent protein. The notable exceptions are for residues in regions of the fragments that form nonnative hydrophobic clusters in the isolated fragments; mutation of the hydrophobic residues involved in these clusters decreases the equilibrium constant for formation of the noncovalent complex less than it does the equilibrium constant for folding of intact protein. The dissociated fragments must be destabilized by mutation of those hydrophobic residues, but to a lesser extent than is the complex itself. These clusters are thus less important energetically in the denatured state of the intact protein. The second-order rate constants for the major phase of association change with mutation, similar results being obtained from fluorescence measurements of the regain of tertiary structure and from circular dichroism measurements of the regain of secondary structure. The rate constants for association correlate well, in general, with the rate constants of refolding of the respective uncleaved proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
MRC Unit for Protein Function and Design, Cambridge University, United Kingdom.
J
J
Ruiz-Sanz
G
G
de Prat Gay
D E
DE
Otzen
A R
AR
Fersht
D002942:Circular Dichroism; D004734:Energy Metabolism; D007700:Kinetics; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary
D010446:Peptide Fragments; D010455:Peptides; D010940:Plant Proteins; C093336:chymotrypsin inhibitor 2