Native Contact Density and Nonnative Hydrophobic Effects in the Folding of Bacterial Immunity Proteins

The bacterial colicin-immunity proteins Im7 and Im9 fold by different mechanisms. Experimentally, at pH 7.0 and 10°C, Im7 folds in a three-state manner via an intermediate but Im9 folding is two-state-like. Accordingly, Im7 exhibits a chevron rollover, whereas the chevron arm for Im9 folding is linear. Here we address the biophysical basis of their different behaviors by using native-centric models with and without additional transferrable, sequence-dependent energies. The Im7 chevron rollover is not captured by either a pure native-centric model or a model augmented by nonnative hydrophobic interactions with a uniform strength irrespective of residue type. By contrast, a more realistic nonnative interaction scheme that accounts for the difference in hydrophobicity among residues leads simultaneously to a chevron rollover for Im7 and an essentially linear folding chevron arm for Im9. Hydrophobic residues identified by published experiments to be involved in nonnative interactions during Im7 folding are found to participate in the strongest nonnative contacts in this model. Thus our observations support the experimental perspective that the Im7 folding intermediate is largely underpinned by nonnative interactions involving large hydrophobics. Our simulation suggests further that nonnative effects in Im7 are facilitated by a lower local native contact density relative to that of Im9. In a one-dimensional diffusion picture of Im7 folding with a coordinate- and stability-dependent diffusion coefficient, a significant chevron rollover is consistent with a diffusion coefficient that depends strongly on native stability at the conformational position of the folding intermediate.Author Summary: In order to fold correctly, a globular protein must avoid being trapped in wrong, i.e., nonnative conformations. Thus a biophysical account of how attractive nonnative interactions are bypassed by some amino acid sequences but not others is key to deciphering protein structure and function. We examine two closely related bacterial immunity proteins, Im7 and Im9, that are experimentally known to fold very differently: Whereas Im9 folds directly, Im7 folds through a mispacked conformational intermediate. A simple model we developed accounts for their intriguingly different folding kinetics in terms of a balance between the density of native-promoting contacts and the hydrophobicity of local amino acid sequences. This emergent principle is extensible to other biomolecular recognition processes.