Domain structure of gpNu1, a phage lambda DNA packaging protein

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• The terminase enzyme complex, comprising gpA and gpNu1 subunits, is essential for bacteriophage lambda's DNA packaging into viral capsids.
• Previous research identified functional domains within gpNu1, including DNA binding, self-association, and gpA interaction regions, but lacked detailed biochemical characterization.

Purpose of the Study:

• To biochemically characterize a deletion mutant of gpNu1 (gpNu1ΔP141) lacking the C-terminal 40 amino acids.
• To investigate the role of these C-terminal residues in gpNu1's DNA binding, self-association, and interaction with the gpA subunit.

Main Methods:

• Cloning, expression, and purification of the gpNu1ΔP141 mutant.
• Circular dichroism (CD) and fluorescence spectroscopy to assess protein folding and stability.
• Analysis of protein aggregation, subunit interactions, and DNA binding affinity and specificity.

Main Results:

• gpNu1ΔP141 retains DNA binding and self-association capabilities but exhibits weak interaction with gpA and fails to form a functional holoenzyme.
• The C-terminal 40 residues are essential for specific DNA binding and proper gpA subunit interaction.
• Spectroscopic studies confirmed that the DNA binding and self-association domains are independent folding units.

Conclusions:

• The C-terminal 40 amino acids of gpNu1 are critical for specific DNA recognition and the formation of a catalytically active terminase holoenzyme.
• These residues mediate essential interactions with the gpA subunit, ensuring correct assembly and function.