The Trp-cage: optimizing the stability of a globular miniprotein

Area of Science:

• Protein Biophysics
• Molecular Biology
• Computational Chemistry

Background:

• The Trp-cage is the smallest miniprotein, making it a model for studying protein folding dynamics.
• The original Trp-cage has a melting point of 42°C but can be stabilized by mutations.
• Previous studies hypothesized specific Pro/Trp interactions are essential for Trp-cage core formation.

Purpose of the Study:

• To investigate the effects of specific mutations on Trp-cage stability and folding pathways.
• To identify key residues and interactions contributing to Trp-cage stabilization.
• To challenge the hypothesis regarding the essential role of Pro/Trp interactions in core formation.

Main Methods:

• Site-directed mutagenesis of the Trp-cage miniprotein.
• Differential scanning calorimetry to measure melting points and protein stability.
• Computational analysis of structural changes and fluxionality.

Main Results:

• Mutations in helical regions yielded a 1.5 kJ/mol fold stabilization per residue replacement.
• Proline mutations at P12 and P18 destabilized the protein by 2.3-3.4 kJ/mol, while P19A destabilized it by 16 kJ/mol.
• A Y3/P19 interaction and Trp burial were identified as crucial for stability, defining an 18-residue folding unit. Stabilizing features include an Arg/Asp salt bridge and a buried H-bonded Ser.

Conclusions:

• Specific Pro/Trp interactions are not essential for Trp-cage core formation.
• The Y3/P19 interaction is a critical stabilizing feature, defining the folding motif.
• Stabilization of the Trp-cage can be achieved through various interactions, including salt bridges and hydrogen bonds.