Studies of asymmetric membrane assembly

Area of Science:

• Biophysics
• Molecular Biology
• Membrane Protein Assembly

Background:

• The M13 bacteriophage major capsid protein integrates into host plasma membranes during infection.
• This protein exhibits specific asymmetric insertion with its amino terminus exposed externally.
• Understanding this process is key to viral assembly and membrane protein dynamics.

Purpose of the Study:

• To investigate the self-assembly mechanism of purified M13 coat protein into synthetic lipid vesicles.
• To elucidate the role of lipid-protein interactions in vesicle formation and protein incorporation.
• To characterize the structural states of M13 coat protein during vesicle assembly.

Main Methods:

• Cholate dilution technique to form phosphatidylcholine vesicles near the lipid's phase transition temperature (Tm).
• Analysis of lipid-protein mixtures using techniques to assess micelle and vesicle formation.
• Chymotrypsin digestion to determine protein structural changes and incorporation into vesicles.

Main Results:

• Lipid in pre-dilution mixtures formed uniform-sized mixed micelles.
• M13 coat protein existed in multiple aggregation states prior to dilution, with no initial lipid interaction.
• Dilution rapidly produced small, closed vesicles with incorporated coat protein (chymotrypsin-resistant).
• Large vesicles formed slowly, maintaining coat protein asymmetry and internal volume.

Conclusions:

• M13 coat protein self-assembly into vesicles is a dilution-dependent process.
• Protein incorporation into vesicles occurs rapidly upon dilution, resulting in a stable, asymmetric structure.
• A model for M13 coat protein-lipid vesicle assembly is proposed, highlighting the importance of lipid micelle intermediates.