Functional and structural properties of cardiotoxin isomers produced by blocking negatively charged groups

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Naja atra cardiotoxin 3 (CTX3) is a protein with known biological activities.
• Specific amino acid residues, like Asp40, Asp57, and Asn60, play crucial roles in protein structure and function.
• Carboxyl groups are key functional groups in amino acids that can influence protein conformation and interactions.

Purpose of the Study:

• To investigate the functional roles of Asp40, Asp57, and C-terminal Asn60 in CTX3 structure and function.
• To understand how modification of these carboxyl groups affects CTX3's conformational properties and membrane interactions.
• To determine the essentiality of specific carboxyl groups for maintaining CTX3's structural topology and biological activity.

Main Methods:

• Chemical modification of carboxyl groups in CTX3 using semicarbazide.
• Analysis of structural changes using circular dichroism (CD) spectroscopy.
• Assessment of membrane permeabilization and leakage induction in phospholipid vesicles and U937 cells.
• Utilized polydiacetylene/lipid assay to study membrane interaction modes.

Main Results:

• Semicarbazide conjugation produced two CTX3 conformational isomers with altered gross and fine structures.
• Carboxyl group modification increased structural flexibility and significantly reduced permeabilizing effects on anionic phospholipid vesicles.
• Modified CTX3 showed reduced membrane leakage activity in U937 cells compared to native CTX3.
• Selective modification of Asp40 did not induce conformational isomerization, indicating Asp57 and Asn60 are critical.

Conclusions:

• The carboxyl groups in Asp57 and Asn60 are essential for maintaining the structural topology of CTX3.
• Modification of carboxyl groups alters the interdependence between CTX3's infrastructure and global conformation.
• These structural changes significantly modulate CTX3's membrane permeabilizing activity, particularly on anionic lipids.