6F1F

The methylene thioacetal BPTI (Bovine Pancreatic Trypsin Inhibitor) mutant structure

Summary for 6F1F

• Entry DOI: 10.2210/pdb6f1f/pdb
• Descriptor: Pancreatic trypsin inhibitor, SULFATE ION, GLYCEROL, ... (4 entities in total)
• Functional Keywords: methylene thioacetal, inhibitor, bpti, methylenedithioether, blood clotting
• Biological source: Bos taurus (Bovine)
• Total number of polymer chains: 5
• Total formula weight: 35285.80

Authors

Lansky, S.,Mousa, R.,Metanis, N.,Shoham, G. (deposition date: 2017-11-21, release date: 2018-07-18, Last modification date: 2024-01-17)

Primary citation

Mousa, R.,Lansky, S.,Shoham, G.,Metanis, N.
BPTI folding revisited: switching a disulfide into methylene thioacetal reveals a previously hidden path.
Chem Sci, 9:4814-4820, 2018

PubMed Abstract: Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue protein that is stabilized by three disulfide bonds at positions 5-55, 14-38 and 30-51. Widely studied for about 50 years, BPTI represents a folding model for many disulfide-rich proteins. In the study described below, we replaced the solvent exposed 14-38 disulfide bond with a methylene thioacetal bridge in an attempt to arrest the folding pathway of the protein at its two well-known intermediates, N' and N*. The modified protein was expected to be unable to undergo the rate-determining step in the widely accepted BPTI folding mechanism: the opening of the 14-38 disulfide bond followed by rearrangements that leads to the native state, N. Surprisingly, instead of halting BPTI folding at N' and N*, we uncovered a hidden pathway involving a direct reaction between the N* intermediate and the oxidizing reagent glutathione (GSSG) to form the disulfide-mixed intermediate N*-SG, which spontaneously folds into N. On the other hand, N' was unable to fold into N. In addition, we found that the methylene thioacetal bridge enhances BPTI stability while fully maintaining its structure and biological function. These findings suggest a general strategy for enhancing protein stability without compromising on function or structure, suggesting potential applications for future therapeutic protein production.

PubMed: 29910933
DOI: 10.1039/c8sc01110a

Experimental method

X-RAY DIFFRACTION (1.716 Å)