The covalent structure of Acanthamoeba actobindin

Area of Science:

• Biochemistry
• Cell Biology
• Protein Science

Background:

• Actobindin is a protein isolated from Acanthamoeba castellanii.
• It exhibits bivalent affinity for monomeric actin.
• This characteristic makes it a potent inhibitor of early actin polymerization.

Purpose of the Study:

• To determine the complete amino acid sequence of actobindin.
• To elucidate the structural basis for its bivalent actin-binding.
• To investigate potential functional similarities with other actin-binding proteins.

Main Methods:

• Protein sequencing using enzymatic and chemical cleavage (trypsin, Staphylococcus V8 protease, endoproteinase Asp-N, CNBr).
• Amino acid sequence analysis to identify repeated segments and conserved motifs.
• Circular dichroism spectroscopy to assess secondary structure content.

Main Results:

• The complete 88-residue amino acid sequence of actobindin was determined.
• Actobindin comprises two nearly identical repeated segments (approx. 33 residues each), accounting for ~76% of the molecule.
• Circular dichroism indicated 15% alpha-helix and 22% beta-sheet structure.
• Sequence analysis revealed similarities to tropomyosin, myosin, paramyosin, alpha-actinin, and profilins.
• Unique sequence similarities were found around trimethyllysine residues compared to elongation factor 1 alpha.

Conclusions:

• The repeated structure of actobindin likely underlies its bivalent actin-binding capability.
• Actobindin shares sequence motifs with various actin-associated proteins, suggesting conserved functional domains.
• Specific sequence similarities around trimethyllysine residues may indicate specialized interactions or post-translational modifications.