I am currently working with liposome deposition of DMPC-DMPG 2:1 on different substrates (glass and equivalent L1 chip of biacore). I am not sure whether I am obtaining fully fused bilayers, I suspect I also have intact liposomes. I am wondering whether the lateral pressure in the liposome bilayer or in the fused bilayer is very different, and of what order of magnitude it can be (32 mN/m?). As a second question, I was wondering whether the lateral pressure in bicelle would be closer to that of liposomes or of fused bilayer (if any difference among them).
The lateral pressure in supported lipid bilayers will vary as a function of area of your chamber (incase you are using a closed chamber). You can easily back-calculate assuming the area of a headgroup to be 0.65 nm2.
If you have fully fused bilayers, you should probably use FRAP (Fluorescence Recovery After Photobleaching) technique to test it by incorporating a fluorescent lipid like NBD-PC or BODIPY-PC. If you get a complete recovery (meaning no immobile fraction), you have fused bialyers.
You can reduce the lipid concentration you use to get rid of intact liposomes. Are you sure they are intact liposomes? Using high salt during fusion can also lead to budding which appear like intact liposomes. You can easily test this preparing your bilayer in lesser salt containing buffer.
I dont have much info regarding bicelles, but i hope this helps. :)
adi
Dear Aditya
Thanks a lot for your response. Actually, I made AFM of the surface I have, and I had liposomes, but it was not clear whether I also had a fused bilayer. It was difficult to do the image because I had to cut the SPR chip... So it would not be so easy to do FRAP either. But still, you mean that from the SPR signal I could calculate back the pressure? The problem is that if I hav both liposomes and bilayer, I will not know what is the lateral pressure. My question is whether there is any difference in the lateral pressure of a liposome or a fused bilayer. In the case of glass I even do not use a chamber when I make the deposition. But I know the total area of the glass slide of course. Still I do not really get how I could calculate back the pressure. The reason I ask is because I have a protein supposed to insert in the bilayer, and I have two binding behaviours, so I was wondering whether the pressure would matterin the binding. If there is a lower pressure in the bilayer, it may facilitate the binding for instance.
There will surely be a difference in the lateral pressure from liposomes and fused bilayers. If you have access to a langmuir apparatus, you could measure the lateral pressure. But unless you have a closed chamber, you can not control the lateral pressure of supported lipid bilayers since the lipids are free to move around. I hope you get what i mean.
I meant that you can calculate your lipid concentration approximately asusming an average head group size of 0.65 nm2. If you know the area of your chamber, you can use avogadro's number to calculate the number of lipid molecules in that area and including volume of your buffer, you will get the lipid concentration. But i realize this will not help you to control lateral pressure.
Lateral pressure heavily influences protein insertion. A high lateral pressure means reduced exposure of hydrophobic acyl chains and vice versa. I think for measuring protein insetion in lipid membranes, langmuir apparatus (LB) is best and i think it can be coupled to an SPR setup too.
Hope this helps! Let me know if you have more queries.
Adi
Ok, so if your substrate is flat (i.e. a bilayer on glass for example) then I would assume little to no pressure really at all. If your bilayer is support via teathers then the suspended regions (depending on teather coverage i.e. the area of the suspended patch) you would have a lateral pressure effect.
Dear Aditya, you are asking a VERY difficult question :). The only configuration in which you can measure the lateral pressure is on a monolayer at the air-water interface, with a Langmuir-Blodgett trough. On the other hand, there is information on the diffusion coefficient of the lipids comparing giant unilamellar vesicles and supported bilayers (on mica). Apparently the interaction with the solid surface diminishes the diffusion quite a lot (3-5 times) in the supported bilayers, probably by a friction effect. (see the works by Hoff, use z-scan FCS as keyword). This is however not the case if you use a polimer cushion or something like that to separate the bilayer from the solid surface. Now the relationship between diffusion coefficient and lateral pressure is not that clear either. Actually there is very little done comparing diffusion of lipids in monolayers and in bilayers. So my answer is... I have no good answer. You will have to study a bit more. Maybe there is some literature on the subject I am not familiar with.
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