Reduced bovine pancreatic trypsin inhibitor has a compact structure

Area of Science:

• Biochemistry
• Protein Folding
• Structural Biology

Background:

• Bovine pancreatic trypsin inhibitor (BPTI) is a well-studied protein.
• Understanding protein folding pathways is crucial for protein engineering and disease research.
• Reduced BPTI (R-BPTI) provides a model for studying protein structure under non-native conditions.

Purpose of the Study:

• To investigate the conformational state of R-BPTI under reducing conditions.
• To determine if R-BPTI adopts a stable structure despite the absence of disulfide bonds.
• To characterize the dynamics and flexibility of R-BPTI.

Main Methods:

• Nonradiative excitation energy transfer (E) measurements.
• Labeling BPTI with donor and acceptor probes at specific lysine residues.
• Utilizing guanidine hydrochloride (Gdn.HCl) and dithiothreitol (DTT) to control protein state.

Main Results:

• High energy transfer efficiencies were observed in R-BPTI, approaching native-state levels.
• Energy transfer efficiency did not decrease with increasing residue separation, contrary to random coil predictions.
• The 1-15 residue segment showed higher energy transfer efficiency in the reduced state than in native or unfolded states, indicating flexibility.

Conclusions:

• R-BPTI exists in a molten globule state under folding, reducing conditions.
• This molten globule state is compact and flexible, with interprobe distances similar to the native state.
• The molten globule structure likely serves as a precursor to the native BPTI structure under oxidizing conditions.