In FRET, we attach a donor and an acceptor molecule and if the donor is in close proximity of the acceptor (with other factors like spectral overlap, etc.), the acceptor gets excited from the energy emitted by the donor and we see fluorescence from the acceptor molecule. This is how it works.
Now suppose we have a protein molecule and we want to compute the distance between two distant amino acids in that protein. So, normally we attach donor to one of the amino acids and an acceptor to another, and see if we are seeing any fluorescence from the acceptor. But what we assume in FRET is that "attaching donor and acceptor molecules will not change the conformation of the protein." What if this is not true? Are our results reliable? Can we cross check?
There are a number of separate things to consider here:
1. Conformation of the Protein: in most cases, attachment of the dye is done after purification, that means with the folded Protein. Therefore, the protein will most likely remain intact (in ist native conformation), assuming mild conditions for the coupling reaction of the fluorophore.
2. A more sincere issue is that dyes (which are often hydrophobic) interact with the protein surface. This alters two parameters: first the quantum yield can be affected (decreased or increased), and secondly the anisotropy of the dyes. This means that for experimental approaches, you should analyse proteins labelled individually with only one dye for exactly these parameters.
3. Given that all these parameters are affected, it may be difficult if not impossible to precisely calculate distances from observed FRET efficiencies. Instead it is more common to calculate changes in FRET Efficiency, for example upon ligand binding or unfolding.
In attached paper is example of FRET between fluorescent protein and conducting polymer.
Article Immunosensor based on fluorescence quenching matrix of the c...
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