Journal articles

The equilibrium and kinetics of folding of the single-domain protein chymotrypsin inhibitor 2 conform to the simple two-state model. The structure of the rate-determining transition state has been mapped out at the resolution of individual side chains by using the protein engineering method on 74 mutants that have been constructed at 37 of the 64 residues. The structure contains no elements of secondary structure that are fully formed. The majority of interactions are weakened by > 50% in the transition state, although most regions do have some very weak structure.

Hammond behavior, in which two neighboring states move closer to each other along the reaction coordinate as the energy difference between them becomes smaller, has previously been observed for the transition state of unfolding of barnase. Here, we report Hammond behavior for the small protein chymotrypsin inhibitor 2 (CI2), which folds and unfolds via a single rate-determining transition state and simple two-state kinetics. Mutants have been generated along the entire sequence of the protein and the kinetics of folding and unfolding measured as a function of concentration of denaturant.

Protein engineering and kinetic experiments indicate that some regions of proteins have partially formed structure in the transition state for protein folding. A crucial question is whether there is a genuine single transition state that has interactions that are weakened in those regions or there are parallel pathways involving many transition states, some with the interactions fully formed and others with the structural elements fully unfolded. We describe a kinetic test to distinguish between these possibilities.

We have prepared a family of peptide fragments of the 64-residue chymotrypsin inhibitor 2, corresponding to its progressive elongation from the N terminus. The growing polypeptide chain has little tendency to form stable structure until it is largely synthesized, and what structures are formed are nonnative and lack, in particular, the native secondary structural elements of alpha-helix and beta-sheet. These elements then develop as sufficient tertiary interactions are made in the nearly full-length chain.

The importance of chaperonin-protein interactions has been investigated by analyzing the refolding of the barley chymotrypsin inhibitor 2 in the presence of GroEL. The chaperonin retards the rate of refolding of wild type and 32 representative point mutants. The retardation of the rate drops to a finite level at saturating concentrations of GroEL, being lowered by a factor of 3-100, depending on the mutation. It is seen qualitatively that truncation of large hydrophobic side chains to smaller side chains weakens binding.

The 64-residue protein chymotrypsin inhibitor 2 (CI2) is a single module of structure. It folds and unfolds as a single co-operative unit by simple two-state kinetics via a single rate determining transition state. This transition state has been characterized at the level of individual residues by analysis of the rates and equilibria of folding of some 100 mutants strategically distributed at 45 sites throughout the protein. Only one residue, a helical residue (Ala16) buried in the hydrophobic core, has its full native interaction energy in the transition state.

There is a region of well-ordered structure in the transition state of folding of chymotrypsin inhibitor 2 (CI2) that consists of N-terminal residues in the unique alpha-helix (residues 12 to 24) plus some long range interactions, in particular those of Ala16 with Ile57 and Leu49 in the hydrophobic core. This is proposed to be a nucleation site. A crucial question for understanding the initiation of protein folding is: when is the nucleation site formed? Is the alpha-helix pre-formed in the nominally unfolded state, or does it require long-range interactions to be stabilized?

Independent experimental and theoretical studies of the unfolding of barley chymotrypsin inhibitor 2 (CI2) are compared in an attempt to derive plausible three-dimensional structural models of the transition states. A very simple structure index is calculated along the sequence for the molecular dynamics-generated transition state models to facilitate comparison with the phi F values. The two are in good agreement overall (correlation coefficient = 0.87), which suggests that the theoretical models should provide a structural framework for interpretation of the phi F values.