Calcium-independent subtilisin by design

Area of Science:

• Structural Biology
• Protein Engineering
• Biochemistry

Background:

• Subtilisin BPN' is a well-studied serine protease.
• Calcium ions play a crucial role in stabilizing the structure and function of many proteins, including subtilisin BPN'.
• Understanding the precise role of calcium binding sites is essential for protein engineering and drug design.

Purpose of the Study:

• To create and characterize a genetically engineered subtilisin BPN' variant lacking the high-affinity calcium site A.
• To investigate the structural and functional consequences of removing calcium site A.
• To explore the utility of this variant for studying protein folding mechanisms.

Main Methods:

• Genetic engineering to produce a subtilisin BPN' mutant with deletion of residues 75-83 (eliminating calcium site A).
• X-ray crystallography to determine the crystal structure of the mutant protein at 1.8 Å resolution.
• Comparison of the structural features of the calcium-depleted mutant with the wild-type protein.

Main Results:

• A continuous helix with normal geometry was observed in the deletion mutant, unlike the interrupted helix in the wild-type protein.
• Residues near the deleted calcium binding loop were repositioned and/or destabilized.
• The absence of the calcium binding loop significantly facilitated protein refolding.

Conclusions:

• The removal of calcium site A in subtilisin BPN' leads to significant structural alterations, including helix restoration.
• The engineered mutant provides a valuable tool for dissecting and analyzing the protein folding process.
• This study represents one of the largest internal protein modifications characterized at atomic resolution.