i am working with a 25 a.a. residue long membrane lytic peptide. I want to do a FRET study so I want to modify its N-terminal to attach a fluorophore. Can anybody tell me whether this is a feasible option.This peptide does not have any tryptophan residue, but a phenylalanine. MD simulation study shows that its N-terminal is involved in membrane interaction. I fear that any modification at N-terminus may change its structure or affect its membrane- interacting capability.
It is certainly feasible to attach a fluorophore to the N-terminal amino group, if it is not blocked, using commercially available amine-reactive dyes. However, if there are lysine residues in the peptide, they will also be labeled. It is challenging to obtain complete selectivity for the amino terminus over lysine. Incorporating the label during solid phase peptide synthesis is an effective way to get selective labeling.
If the N-terminus is involved in the function of the peptide, it is almost certain that modifying it with a fluorophore will alter its function. It would be advisable to attach the fluorophore to a position that is not influential in function. Even then, you will still have to assess whether the modification has altered the function.
Due to the potential difficulties of using a covalently attached dye in your experiment, have you considered using a dye that non-covalently binds your protein? You should be able to get good results with the right fluorophore.
Thanks a lot Adam for your response and for the time you invested. However my peptide doesn't have any lysine residue in it. So labeling that is out of the picture. MD simulation studies does show the involvement of N-terminus in membrane interaction.
I was planning to use IANBD as the fluorophore but that requires a cysteine residue. And as you already mentioned any modification at N-terminal may alter its structure and affect its function which was exactly y thought, I am afraid I don't have the liberty to make change at any other position as the N-terminus coil area is immediately followed by a helical structure.
The wild type structure of the peptide presents a helix-turn-helix motif. I tried the secondary structure prediction of the peptide by doing various mutations (by adding lysine and cysteine) and every time I find the structure being changed drastically.
I am not sure my approach is right or should I try other technique like NMR to address my problems.
Thank you so much Andrew for your response and time. That is a good suggestion. I will definitely look into it and try to find a way out.
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