I am trying to determine the orientation of a small peptide (
Do you mean that you get beta sheet structure because your protein aggregated in Methanol?
Can you dry your plate to let lipid form bilayer 1st and then add buffer dissolved peptide? in this way the peptide can only have one orientation.
Cydi from EZBiolab
Is it an alpha helix in solution with or without liposomes? If you have measured an isotropic CD signal in the absence of lipids, and the conformation in the presence of lipids is not known, I think it is worth your while to check the isotropic CD signal with unilamellar vesicles and lipid refractive index-matched buffer (to reduce scattering at low wavelengths) to see if the lipids induce alpha helix to beta sheet transition (especially anionic lipids can do that). In any case, it is probably better that you prepare multilamellar vesicles or small unilamellar vesicles (in buffer) from your lipids, then add the protein, and dry this, or first dry the aqeous lipid solution, then add your protein. The aqueous solution makes the drying part a bit more tedious, though, and you may want to use gas devoid of oxygen for the drying and, if necessary, lyophilization. You can get bilayer stacks pretty easily from pretty much any bilayer lipid when drying them. After all, continuous lamellar phase tends to be the thermodynamically stable phase, not the liposomal phase. Large bilayer stacks are used for work with ATR-FTIR. Search for example Erik Goormaghtigh and bilayer stacks, and you can find methods for preparing stacks of ~100 bilayers.
Thank you for the responses.
Cydi, yes, the protein aggregates in methanol. I tried drying the lipid first, but when I added the peptide after I got large aggregates on the slide.
Juha-Matti I hadn't thought of the lipids inducing helix-to-beta transition. I will look into this. Also, thank you for the reference. I am trying to dry the aqueous vesicle solution now, however the resulting film is very imperfect. Do you know of a good method to dry it homogeneously?
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