Intermediates and the folding of proteins L and G

We use a minimalist protein model, in combination with a sequence design strategy, to determine differences in primary structure for proteins L and G, which are responsible for the two proteins folding through distinctly different folding mechanisms. We find that the folding of proteins L and G are consistent with a nucleation‐condensation mechanism, each of which is described as helix‐assisted β‐1 and β‐2 hairpin formation, respectively. We determine that the model for protein G exhibits an early intermediate that precedes the rate‐limiting barrier of folding, and which draws together misaligned secondary structure elements that are stabilized by hydrophobic core contacts involving the third β‐strand, and presages the later transition state in which the correct strand alignment of these same secondary structure elements is restored. Finally, the validity of the targeted intermediate ensemble for protein G was analyzed by fitting the kinetic data to a two‐step first‐order reversible reaction, proving that protein G folding involves an on‐pathway early intermediate, and should be populated and therefore observable by experiment.