It has been seen in several proteins that functional residues affect folding.

Using folding simulations of ecoRNase-H, we find that substrate binding residues determine the protein folding mechanism.

A set of aromatic residues are clustered together (“specifically packed”) in a region implicated in substrate binding in ecoRNase-H.

Their packing makes this region fold early and determines the folding route.

Investigating the molecular basis of folding route choice can help understand how proteins accommodate both their function and their folding within a single amino acid sequence.