The nucleation-collapse mechanism in protein folding: evidence for the non-uniqueness of the folding nucleus

Abstract

Background: Recent experimental and theoretical studies have shown that several small proteins reach the native state by a nucleation-collapse mechanism. Studies based on lattice models have been used to suggest that the critical nucleus is specific, leading to the notion that the transition state may be unique. On the other hand, results of studies using off-lattice models show that the critical nuclei should be viewed as fluctuating mobile structures, thus implying non-unique transition states.

Results: The microscopic underpinnings of the nucleation-collapse mechanism in protein folding are probed using minimal off-lattice models and Langevin dynamics. We consider a 46-mer continuum model which has a native β-barrel-like structure. The fast-folding trajectories reach the native state by a nucleation-collapse process. An algorithm based on the self-organized neural nets is used to identify the critical nuclei for a large number of rapidly folding trajectories. This method, which reduces the determination of the critical nucleus to one of ‘pattern recognition’, unambiguously shows that the folding nucleus is not unique. The only common characteristics of the mobile critical nuclei are that they are small (containing on average 15–22 residues) and are largely composed of residues near the loop regions of the molecule. The structures of the transition states, corresponding to the critical nuclei, show the existence of spatially localized ordered regions that are largely made up of residues that are close to each other. These structures are stabilized by a few long-range contacts. The structures in the ensemble of transition states exhibit a rather diverse degree of similarity to the native conformation.

Conclusions: The multiplicity of delocalized nucleation regions can explain the two-state folding by a nucleation-collapse mechanism for small single-domain proteins (such as chymotrypsin inhibitor 2) and their mutants. Because there are many distinct critical nuclei, we predict that the folding kinetics of fast-folding proteins will not be drastically changed even if some of the residues in a ‘typical’ nucleus are altered.