Interplay between drying and stability of a TIM barrel protein: a combined simulation-experimental study

Area of Science:

• Protein biophysics
• Computational biology
• Structural biology

Background:

• Nanoscale dewetting is increasingly recognized for its influence on protein stability, function, and folding dynamics.
• Hydrophobic amino acid clusters within proteins are potential sites for water exclusion (dewetting).

Purpose of the Study:

• To investigate the role of nanoscale dewetting in the stability and folding of the alpha-T-Sarcin (αTS) TIM barrel protein.
• To validate the hypothesis that drying within hydrophobic clusters affects protein structure and stability using a combined simulation-experimental approach.

Main Methods:

• Molecular dynamics simulations to observe water density fluctuations and dewetting transitions in isoleucine, leucine, and valine (ILV) clusters.
• Site-directed mutagenesis, substituting residues with alanine or asparagine to probe the effects on dewetting and protein stability.
• Experimental characterization of protein stability, secondary structure, and compactness for wild-type and mutant variants.

Main Results:

• Simulations revealed intermittent dewetting in ILV clusters within the αTS TIM barrel protein.
• Mutations to less hydrophobic residues (alanine) weakened dewetting, with effects varying by mutation site.
• Replacing buried leucines with polar asparagines enhanced wetting in N- and C-terminal clusters.
• Experimental data for asparagine variants correlated with simulated preferential drying of the N-terminal cluster in intermediate states.
• Unexpectedly, mutations revealed alternative stable folds within the native protein basin.

Conclusions:

• Drying within hydrophobic ILV clusters is critical for the folding and stability of the αTS TIM barrel protein.
• The study provides experimental validation for the role of nanoscale dewetting in protein biophysics.
• The findings suggest that controlling dewetting in hydrophobic clusters could be a strategy for protein engineering.