Small Proteins Fold Through Transition States With Native-like Topologies

The folding pathway of common-type acyl phosphatase (ctAcP) is characterized using ψ-analysis, which identifies specific chain–chain contacts using bi-histidine (biHis) metal-ion binding sites. In the transition state ensemble (TSE), the majority of the protein is structured with a near-native topology, only lacking one β-strand and an α-helix. ψ-Values are zero or unity for all sites except one at the amino terminus of helix H2. This fractional ψ-value remains unchanged when three metal ions of differing coordination geometries are used, indicating this end of the helix experiences microscopic heterogeneity through fraying in the TSE. Ubiquitin, the other globular protein characterized using ψ-analysis, also exhibits a single consensus TSE structure. Hence, the TSE of both proteins have converged to a single configuration, albeit one that contains some fraying at the periphery. Models of the TSE of both proteins are created using all-atom Langevin dynamics simulations using distance constraints derived from the experimental ψ-values. For both proteins, the relative contact order of the TS models is ∼80% of the native value. This shared value viewed in the context of the known correlation between contact order and folding rates, suggests that other proteins will have a similarly high fraction of the native contact order. This constraint greatly limits the range of possible configurations at the rate-limiting step.