Small proteins fold through transition states with native-like topologies

Area of Science:

• Protein folding dynamics
• Biophysics
• Structural biology

Background:

• Understanding protein folding pathways is crucial for deciphering biological function and disease mechanisms.
• The transition state ensemble (TSE) represents a critical, yet transient, phase in protein folding.
• Psi-analysis offers a method to probe protein chain-chain contacts within the TSE.

Purpose of the Study:

• To characterize the folding pathway of common-type acyl phosphatase (ctAcP) using psi-analysis.
• To investigate the structural properties of the transition state ensemble (TSE) for ctAcP and ubiquitin.
• To model the TSE structures and assess their contact order in relation to folding rates.

Main Methods:

• Psi-analysis utilizing bi-histidine (biHis) metal-ion binding sites to identify chain-chain contacts.
• Characterization of fractional psi-values to detect microscopic heterogeneity in the TSE.
• All-atom Langevin dynamics simulations constrained by experimental psi-values to model TSE structures.

Main Results:

• The TSE of ctAcP is largely structured, resembling native topology but lacking a beta-strand and an alpha-helix.
• A fractional psi-value at the amino terminus of helix H2 indicates peripheral fraying in the ctAcP TSE.
• Both ctAcP and ubiquitin exhibit a single consensus TSE structure with approximately 80% of the native contact order.

Conclusions:

• The transition state ensemble for both ctAcP and ubiquitin converges to a single configuration, with some peripheral fraying.
• The high, conserved relative contact order in the TSE models suggests a general constraint on the rate-limiting step of protein folding.
• This finding implies that other proteins likely share a similarly high fraction of native contact order in their rate-limiting folding step.