Conformational changes in bacteriophage phi 29 connector prevents DNA-binding activity

Area of Science:

• Molecular Biology
• Virology
• Structural Biology

Background:

• Bacteriophage phi 29 utilizes a sophisticated DNA packaging mechanism.
• The connector protein p10 is essential for this process.
• Understanding p10's role is key to deciphering viral DNA translocation.

Purpose of the Study:

• To investigate the role of the connector protein p10's chemical integrity in bacteriophage phi 29 DNA packaging.
• To determine the impact of proteolytic cleavage on p10 function and structure.
• To map the DNA-binding domain of the connector protein.

Main Methods:

• In vitro DNA packaging assays using a defined system.
• Proteolytic cleavage of p10 and isolated connectors.
• Electron microscopy for structural analysis.
• Analytical ultracentrifugation and low-angle X-ray scattering.
• Amino acid analysis to identify cleavage sites.

Main Results:

• Proteolytic cleavage of p10 inactivated in vitro DNA packaging.
• Cleaved connectors lost DNA-binding activity without significant changes in overall shape or size.
• Proteolysis resulted in loss of amino acids from both termini and structural reorganization of the connector.
• Specific cleavage sites were identified, aiding in the localization of the DNA-binding domain.

Conclusions:

• The chemical integrity of bacteriophage phi 29 connector protein p10 is crucial for DNA packaging.
• The N- and C-terminal regions of p10 are involved in DNA binding and/or packaging regulation.
• Proteolysis-induced structural changes in p10 directly impair its DNA packaging function.