Refinement of the three-dimensional solution structure of barley serine proteinase inhibitor 2 and comparison with the structures in crystals

159

153

Previous experimental and theoretical studies have produced highresolution descriptions of the native and folding transition states of chymotrypsin inhibitor 2 (CI2). In similar fashion, here we use a combination of NMR experiments and molecular dynamics simulations to examine the conformations populated by CI2 in the denatured state. The denatured state is highly unfolded, but there is some residual native helical structure along with hydrophobic clustering in the center of the chain. The lack of persistent nonnative structure in the denatured state reduces barriers that must be overcome, leading to fast folding through a nucleation-condensation mechanism. With the characterization of the denatured state, we have now completed our description of the folding͞ unfolding pathway of CI2 at atomic resolution.CI2 ͉ nuclear magnetic resonance ͉ molecular dynamics simulations ͉ conformational transitions ͉ nucleation-condensation P rotein folding is a rapid and complex process that is difficult to characterize. To add to this difficulty, the denatured state consists of a large ensemble of conformations interconverting at a rapid rate. The denatured state is often assumed to be devoid of intramolecular interactions, such that the stability of a protein can be explained purely in terms of interactions in the native state. In recent years, it has become apparent that many proteins contain residual structure in the denatured state (ref. 1 and refs. therein). However, detailed characterization of this structure is very challenging, if not impossible in many cases. As such, during folding one follows the transition of a diverse system from an unknown starting point to a well-ordered native state. Further information about the diversity, dynamics, and structure of the denatured state is necessary to characterize and understand better this process.The simplest folding pathway to define is two state, i.e., involving only the denatured and native states, which are separated by the energetically unfavorable transition state. Chymotrypsin inhibitor 2 (CI2) was the first protein shown to fold by a two-state mechanism, and it has since been the focus of a number of experimental and theoretical studies. It is a 64-residue protein that consists of an ␣-helix and a three-stranded ␤-sheet (Fig. 1). The main hydrophobic core is formed by the packing of the ␣-helix against the ␤-sheet.Experimentally, the structure of the transition state has been studied by the protein engineering (⌽-value) method (2). In combination with molecular dynamics (MD) simulations, an atomic-resolution model of the transition state has been proposed (3-7) and verified (8). The rate-limiting step for the folding of CI2 involves the final expulsion of water molecules from the exposed nonpolar side chains and the tight packing of the hydrophobic core. The transition state is similar to an expanded native state with some disruption of the secondary structure.In contrast, the denatured state of CI2 appears to be largely unstructured as probed by NMR studies of variou...

mentioning

confidence: 99%

Protein folding from a highly disordered denatured state: The folding pathway of chymotrypsin inhibitor 2 at atomic resolution

Kazmirski

Wong

Freund

et al. 2001

159

153

“…The structure of the first 19 residues is not resolved by either x-ray crystallography (10) or two-dimensional NMR studies (11)(12)(13)(14)(15). The structure of a pseudo-wild-type form of C12 in which these residues have been omitted has recently been solved to 1.7-A resolution ( Fig.…”

mentioning

confidence: 99%

Characterization of the transition state of protein unfolding by use of molecular dynamics: chymotrypsin inhibitor 2.

Daggett

1994

238

212

Temperature-induced unfolding of chymotrypsin inhibitor 2 in water was investigated by molecular dynamics smulations. (6,9). These studies suggest that the edges of the core are significantly weakened in the transition state, while the center of the core remains partially intact. One particular residue, Val-38, is special in that it is more buried in the transition state than it is in the native state.

“…It is small and monomeric. Its three-dimensional structure has been solved in both the crystal (26,27) and the solution states (28)(29)(30)(31). Despite its small size, there is considerable secondary and tertiary structure present and a small, compact hydrophobic core.…”

mentioning

confidence: 99%

Structure of the transition state for the folding/unfolding of the barley chymotrypsin inhibitor 2 and its implications for mechanisms of protein folding.

Otzen

Itzhaki

elMasry

et al. 1994

216

181