This study investigates the interaction between a phospholipid-binding protein and a lipid bilayer using dissipative microgravimetry. The method involves monitoring changes in frequency and dissipation as vesicles interact with the sensor surface and the target protein binds to the lipid bilayer.
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.
Carefully dock the plasma-cleaned sensors into the four flow chambers using tweezers. Avoid any pressure on or torsion of, the chambers and tubes that may cause leaking. Flush the system with citrate buffer in the open flow mode for 10 minutes.
Launch the program. Start recording any changes in the frequency and dissipation of the first fundamental tone and overtones using the software until the frequency and dissipation baselines are stable.
When the baselines are stable, apply the SUV suspension in citrate buffer. Using a reaction vessel, remove 1.5 milliliters of the dead volume. Then, close the system in the loop flow mode. Record the frequency dissipation shift for another 10 minutes.
When the SLB is stable, equilibrate the system with the running buffer at the required calcium concentrations in an open-flow mode for 40 minutes. Add the protein to the running buffer containing calcium. Perform the application of the protein in a loop-flow mode until an equilibrium steady state is reached.
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