Consequences of localized frustration for the folding mechanism of the IM7 protein

Abstract

In the laboratory, IM7 has been found to have an unusual folding mechanism in which an "on-pathway" intermediate with nonnative interactions is formed. We show that this intermediate is a consequence of an unusual cluster of highly frustrated interactions in the native structure. This cluster is involved in the binding of IM7 to its target, Colicin E7. Redesign of residues in this cluster to eliminate frustration is predicted by simulations to lead to faster folding without the population of an intermediate ensemble.

Fig. 1.

Characterization of the IM7 wild type folding thermodynamics and its intermediate features. (A) Free energy (in units of ε) of the IM7 at T̄ = 1.0 as a function of Q_W and rmsd. The wells correspond to the unfolded (U), intermediate (I), and native (N) states. (B) The average fraction of nonnative contacts as a function of the rmsd. In the red curve, we consider only the interactions between the first half of the protein (residues 1–46) with the fourth helix (residues 68–86). The blue curve shows the same ratio but only for the contacts internal to the helix III region (residues 47–67), and the black curve represents the contacts between the first half and the helix III region. Also displayed with vertical dotted lines are the boundaries used to define the intermediate state. (C) The percentage of the transitions observed between the three states U, I, and N suggests the intermediate to be on-pathway in the folding reaction. The states are operatively defined in terms of Q_W and rmsd as U = [0.43; 0.47] × [8.4; 9.7], I = [0.45; 0.51] × [5.7; 7.0] and U = [0.55; 0.60] × [2.5; 3.3], and a transition is counted every time the trajectory in Q_W, rmsd space leaves one state and enters another without passing through the third. The percentage of jumps are calculated out of 1,890 observed transitions during 40 independents simulations at T̄ = 1.0. Three representative snapshots of each state also are shown, with the helix III region highlighted in yellow.

Fig. 2.

Native-state residual frustration and two-state behavior of the designed double mutant Y55R, Y56N. (A) Local frustration is depicted on the native IM7 wild-type structure (from Protein Data Bank ID code 1AYI). A large cluster of minimally frustrated contacts (green) defines the core of the protein, but some highly frustrated contacts (F_ij < −1) surround the core (indicated in red). (B) Native contacts of wild-type IM7 in the native (upper-left) and intermediate (lower-right) states. Highly frustrated contacts (red) are primarily local compared with all contacts present in the native state. Within region III (blue box), multiple frustrated contacts remain unformed in the intermediate. (C) Free energy (in units of ε) of the IM7 designed mutant at T̄ = 1.0 as a function of Q_W and rmsd. The minima correspond well to the unfolded (U) and native (N) states of the wild-type protein.

Fig. 3.

Specific intermediate state redesign. (A) Local frustration is depicted on a selected IM7 intermediate structure. Minimally frustrated contacts present in the crystal structure (green) are distinguished from those that are nonnative (blue). A distinct nonnative cluster can be observed involving interactions between helix IV and the helix I–II region. Native (red) and nonnative (orange) frustrated contacts surround the core. (B) Two clusters of minimally frustrated contacts (blue) characterize the nonnative contacts in the intermediate state (lower-right). The wild-type native-state contacts also are shown (upper-left). (C) Free energy (in units of ε) of the IM7 redesigned mutant K20Y, N79Y at T̄ = 1.0 as a function of Q_W and rmsd. (Inset) The free energy as a function of Q_W to the crystal structure and Q_W to a representative structure from the wild-type intermediate basin.