Dear Scientist,
My actual task is to incorporate some membranes proteins (multipass, 7TM, singlepass) into liposomes and nanodiscs.
Native membranes are polarized structures with different lipid composition. In liposomes and nanodiscs this state is not possible to obtain so I have to make decision which lipids or lipid mixtures I am going to use as a medium for my proteins.
I wanted to ask if anybody has some experience in protein incorporation into lipid structures.
Do you know any database describing lipid-protein interactions? How do I know If I should use pure lipids or natural extracts an what kind?
First thing that comes to my mind is protein pI. Should I pick negatively charged membrane for proteins with pI>8 and neutral one for acidic proteins ?
I would be grateful for any help.
Avanti lipids is a good place to start. Very useful web site and helpful staff. I used E. coli phospholipids for reconstituting MsbA (an E. coli protein-6TM). The method is described fully in my 2002 paper (JBC 277, 36697). Good luck!
I bought lipid from Avanti Lipids as well. But I am working with ENaC. I am trying to incorporate ENaC in lipid mixture. See me review paper, where I summarized different papers and they used different protein-lipid interaction.
http://www.mdpi.com/1422-0067/14/11/21561
Usually it takes time to incorporate protein in lipids. If you already have Some researchers apply -40mV, and this will accelerate the procedure. Are you trying to measure ion-channel activity of 7TM?
Dear Muhammad,
Our incorporation is for other purposes. We need it for structural studies and phage display. Problem is that I have small amounts of my proteins so it is not possible to test different lipid mixtures. I have to choose them very wisely not to lose my targets.
I have some lipids from Avanti e.g. E.coli polar lipids, egg PC, POPC, DMPC, sphingomyelin, DOPC a probably few more.
I just want to know if there are any general guidelines for choosing lipids.
Let's take an example of bacteriorhodopsin, 7TM protein, pI~5.5. Do you think I can use E.coli polar lipids, which are composed majorly of PE, but also have a lot of negatively charged PG and cardiolipin? Or maybe I should use egg PC with neutral polar groups? I may assume that negatively charged protein will repel negatively charged lipids and it can affect its stability. Did you meet this problem, anyone?
It seems really tricky. I think you have to look at Williams paper as he stated that he used to work with E.Coli phospholipids with protein 6TM.
William, thank you for your publication. As i can see you used E.coli polar lipids. According to Avanti's page this mixture has negative net charge and MsbA protein in charged positively(P60752). One of my targets is bacterial ion channel, pI=5.1 I am affraid that E.coli polar extract might not be the best choice for this one. I am thinking about using mixture of this one and egg PC.
What is the natural composition of the membrane where your protein belongs? Going from there, you can try to mimic the best as you can this composition. In lipid world, the simplest the better. DOPC/DPPC with 10/20 % cholesterol and DPPS 1:10 can be a good starting point. The best method i've tried for protein incorporation into a membrane is reverse micelle with ether evaporation or Octyl glucoside detergent exchange. Consult the paper about (bacterio) rhodopsin incorporation into liposome. Choose your lipids in regards of Tm and lipid phase. Finally, be patient, these processus can take several days. All depend on what technique or measurements you're gonna use afterward: NMR, Fluorescence, electron microscopy...Good luck!
Thanks for your advice Danny.
I have three different proteins, two isoforms of each. They are mostly channels, bacterial or human. Now I am trying to insert them into nanodiscs
I started with mixture of E.coli polar lipids with eggPC and it didn't work too well. Proteins have incorporated but majority precipitated during upconcentration. My second approach is eggPC with POPG. Now it looks better. I have some precipitate but a lot more in solution. And the gel filtration shows proper profiles. It might be true, that easier is better for this case.
You can also have a look on bicelles (DHPC/DPPC) which are useful in nmr spectroscopy for membrane protein. I don't know what's your 'recipe' for the proteins incorporation but if it include an ether evaporation step, this one must be done thoroughly and very slowly above the Tm of your lipid mixture...all is about lipid phases.
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