Dissipative Microgravimetry Technique to Study Protein-Lipid Bilayer Interaction
Transcrição
To study the interaction between a phospholipid-binding protein and a lipid bilayer through dissipative microgravimetry, take a microbalance with a quartz sensor. Upon applying a suitable voltage, the quartz layer — sandwiched between two metal electrodes — oscillates at a specific frequency.
Add a suspension of small unilamellar vesicles — consisting of a single lipid bilayer — onto the sensor. The vesicles adsorb on the silica-coated hydrophilic surface — increasing the sensor mass and proportionally decreasing its oscillation frequency.
The buffer-filled vesicles act as a viscoelastic layer, leading to dissipation — the dampening of the oscillation. The adsorbed vesicles rupture, releasing the enclosed buffer. The resultant decrease in mass increases the oscillation frequency.
The ruptured vesicles form a continuous bilayer, mimicking a biological membrane. The rigidity of the bilayer decreases the dissipation.
Add a buffer containing calcium ions, along with the target protein. The calcium ions bind to the protein and change its conformation — enabling binding to the lipid molecules in the bilayer.
Protein binding increases the mass of the sensor — leading to a decreased frequency. The structural integrity of the bilayer remains unaffected — causing only a slight increase in the dissipation.
Add a chelating agent to chelate the calcium ions — dissociating the proteins from the bilayer.
The absence of bound proteins returns the frequency and dissipation of the oscillation to the non-protein-bound levels — confirming that the binding is solely calcium-dependent and that the bilayer remains intact.
