Structural Characterization of the Early Events in the Nucleation–Condensation Mechanism in a Protein Folding Process

The
nucleation–condensation mechanism represents a major
paradigm to understand the folding process of many small globular
proteins. Although substantial evidence has been acquired for this
mechanism, it has remained very challenging to characterize the initial
events leading to the formation of a folding nucleus. To achieve this
goal, we used a combination of relaxation dispersion NMR spectroscopy
and molecular dynamics simulations to determine ensembles of conformations
corresponding to the denatured, transition, and native states in the
folding of the activation domain of human procarboxypeptidase A2 (ADA2h).
We found that the residues making up the folding nucleus tend to interact
in the denatured state in a transient manner and not simultaneously,
thereby forming incomplete and distorted versions of the folding nucleus.
Only when all the contacts between these key residues are eventually
formed can the protein reach the transition state and continue folding.
Overall, our results elucidate the mechanism of formation of the folding
nucleus of a protein and provide insights into how its folding rate
can be modified during evolution by mutations that modulate the strength
of the interactions between the residues forming the folding nucleus.