Phi-values for BPTI folding intermediates and implications for transition state analysis.

Area of Science:

• Biochemistry
• Protein folding dynamics
• Structural biology

Background:

• Bovine pancreatic trypsin inhibitor (BPTI) is a model system for studying protein folding.
• Understanding protein folding intermediates is crucial for comprehending the overall folding process.

Purpose of the Study:

• To investigate the structural characteristics of two key intermediates in the BPTI folding pathway.
• To determine the stability and conformational properties of these intermediates using amino acid replacements.

Main Methods:

• Utilized amino acid replacements to probe 14 specific sites within BPTI folding intermediates.
• Employed Nuclear Magnetic Resonance (NMR) spectroscopy to analyze the conformational states of the intermediates.
• Calculated phi-values to assess the stability of native protein relative to intermediates.

Main Results:

• One intermediate (30--51 disulfide) exhibited a native-like conformation in approximately two-thirds of its structure.
• A second intermediate (30--51 and 5--55 disulfides) possessed a fully folded conformation.
• Phi-values <1 for most residues indicated significantly greater destabilization of the native protein compared to intermediates.

Conclusions:

• Protein folding intermediates and transition states may possess more structure than previously inferred from phi-value analysis alone.
• The observed high degree of structure in BPTI intermediates challenges existing models of protein folding.
• These findings necessitate a re-evaluation of how protein folding pathways are typically represented.