Trapping the On-pathway Folding Intermediate of Im7 at Equilibrium

The four-helical protein Im7 folds via a rapidly formed on-pathway intermediate (kUI=3000 s−1 at pH 7.0, 10 °C) that contains three (helices I, II and IV) of the four native α-helices. The relatively slow (kIN=300 s−1) conversion of this intermediate into the native structure is driven by the folding and docking of the six residue helix III onto the developing hydrophobic core. Here, we describe the structural properties of four Im7* variants designed to trap the protein in the intermediate state by disrupting the stabilising interactions formed between helix III and the rest of the protein structure. In two of these variants (I54A and L53AI54A), hydrophobic residues within helix III have been mutated to alanine, whilst in the other two mutants the sequence encompassing the native helix III was replaced by a glycine linker, three (H3G3) or six (H3G6) residues in length. All four variants were shown to be monomeric, as judged by analytical ultracentrifugation, and highly helical as measured by far-UV CD. In addition, all the variants denature co-operatively and have a stability (ΔGUF) and buried hydrophobic surface area (MUF) similar to those of the on-pathway kinetic intermediate. Structural characterisation of these variants using 1-anilino-8-napthalene sulphonic acid (ANS) binding, near-UV CD and 1D 1H NMR demonstrate further that the trapped intermediate ensemble is highly structured with little exposed hydrophobic surface area. Interestingly, however, the structural properties of the variants I54A and L53AI54A differ in detail from those of H3G3 and H3G6. In particular, the single tryptophan residue, located near the end of helix IV, and distant from helix III, is in a distinct environment in the two sets of mutants as judged by fluorescence, near-UV CD and the sensitivity of tryptophan fluorescence to iodide quenching. Overall, the results confirm previous kinetic analysis that demonstrated the hierarchical folding of Im7 via an on-pathway intermediate, and show that this species is a highly helical ensemble with a well-formed hydrophobic core. By contrast with the native state, however, the intermediate ensemble is flexible enough to change in response to mutation, its structural properties being tailored by residues in the sequence encompassing the native helix III.