A folding nucleus and minimal ATP binding domain of Hsp70 identified by single-molecule force spectroscopy

Significance Proteins with a similar structure can have largely different folding properties. Although some fold readily, others can only assume their native structure through the help of chaperone proteins. Partially folded intermediates play a key role in defining those folding differences. However, owing to their transient nature, they are not amenable to the structural investigation. Using a combination of single-molecule mechanics, protein engineering, and crystallography, we identified a stable native-like functional nucleus, which is a critical intermediate for spontaneous folding of the Hsp70 nucleotide-binding domain. Based on our findings, we engineered a chimera turning a homologous but folding-incompetent protein into a spontaneously folding protein that is enzymatically active. Our results have implications for the folding of actin from the same superfamily.