Subtilisin-cleaved actin: polymerization and interaction with myosin subfragment 1

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Actin is a crucial protein in muscle and non-muscle cells, forming filaments essential for cell structure and motility.
• Understanding actin's structure-function relationship is key to deciphering its diverse cellular roles.

Purpose of the Study:

• To investigate the functional consequences of specific actin cleavage on its polymerization and interactions.
• To elucidate the role of the N-terminal and C-terminal regions in actin's macromolecular associations.

Main Methods:

• Homogeneous G-actin was proteolyzed using subtilisin at a controlled enzyme-to-actin ratio.
• Cleavage site was identified via sequence analysis.
• Polymerization kinetics, critical concentrations, and interactions with myosin subfragment 1 (S-1) and DNase I were assessed under nondenaturing conditions.

Main Results:

• Subtilisin cleaved actin into 9-kDa (N-terminal) and 36-kDa (C-terminal) fragments, associated under nondenaturing conditions.
• Cleaved actin exhibited slower polymerization rates and higher critical concentrations compared to intact actin.
• Actin affinity for S-1 decreased 10-fold, while S-1 ATPase activity (Vmax) remained unchanged; DNase I affinity was also reduced.

Conclusions:

• Subtilisin-induced cleavage significantly alters actin's polymerization dynamics and its interactions with regulatory proteins.
• These findings highlight the importance of the cleaved peptide regions in maintaining actin's native functional properties.
• The study provides insights into actin's structural basis for macromolecular interactions.