Phi-value analysis by molecular dynamics simulations of reversible folding

Area of Science:

• Biophysics
• Computational Biology
• Protein Science

Background:

• Phi-value analysis compares mutation effects on folding kinetics and thermodynamic stability to probe protein-folding transition states (TS).
• Understanding the protein-folding TS is crucial for predicting protein structure and function.

Purpose of the Study:

• To investigate the structure of the protein-folding TS using molecular dynamics (MD) simulations on single-point mutants.
• To assess the reliability and limitations of Phi-value analysis in characterizing TS structures.

Main Methods:

• Performed 0.65 ms of MD simulations for a 20-residue antiparallel beta-sheet peptide and its single-point mutants.
• Sampled 57-120 folding events per mutant to accurately estimate folding/unfolding rates and stability changes.
• Calculated Phi values from MD trajectories and analyzed TS structures, including nonnative interactions.

Main Results:

• Phi values are reliable when stability loss exceeds ~0.6 kcal/mol (observed in 8/32 mutants).
• Mutated peptides exhibited TS heterogeneity, similar to the wild type, suggesting multiple folding pathways.
• Single-point mutations induced TS shifts not always detected by Phi-value analysis.
• Specific nonnative interactions were observed at the TS in most peptides.

Conclusions:

• Phi-value analysis is a valuable tool but can overestimate TS nativeness when nonnative interactions are neglected.
• MD simulations provide detailed insights into TS structure and folding pathways, complementing traditional Phi-value analysis.
• Protein folding is complex, involving heterogeneous transition states and potential nonnative interactions.