During literature research, I have come across articles describing the use of fluorescent tagged proteins/drugs/peptides to investigate ligand binding in an FPS experiment, while protein auto fluorescence appears to be applied to study protein folding/aggregation. I am wondering about the concerns of trying to assess ligand binding using the natural fluorescence of a protein. Is it possible that the main issue would occur with the experimental sensitivity of this experiment? Any literature references would be very much appreciated.
Intrinsic fluorescence titrations are a clean way for monitoring ligand binding, all the more so when the ligand is not fluorescent. Fluorescence intensity is weak, but usually suitable to that purpose, provided that the absorbance of the protein sample is lower than 0.1 at the excitation wavelength. If you fix this at 280 nm, the signal will be stronger, as it arises from both tryptophan and tyrosine and to a lesser extent from phenylalanine. You can also monitor mostly tryptophan emission at the cost of a lower signal by fixing the excitation wavelength at 295 nm. The fluorescence intensity can either increase or decrease upon binding, depending on the protein. Information on how to perform calculations to obtain the binding constant for 1:1 binding can be found here: https://www.researchgate.net/publication/8065951_Receptor_fragment_approach_to_the_binding_between_CCK8_peptide_and_cholecystokinin_receptors_a_fluorescence_study_on_type_B_receptor_fragment_CCK(B)-R_(352-379)?ev=prf_pub.
Exhaustive information on fluorescence can be found in http://www.springer.com/chemistry/analytical+chemistry/book/978-0-387-31278-1.
Article Receptor fragment approach to the binding between CCK8 pepti...
It seems to me that protein autofluorescence would be quite weak compared with the fluorescence of a tag or fluorophore. In addition, the emission peak of the autofluorescence might be incompatible with the emission of your ligand or fluorophore.
Intrinsic fluorescence titrations are a clean way for monitoring ligand binding, all the more so when the ligand is not fluorescent. Fluorescence intensity is weak, but usually suitable to that purpose, provided that the absorbance of the protein sample is lower than 0.1 at the excitation wavelength. If you fix this at 280 nm, the signal will be stronger, as it arises from both tryptophan and tyrosine and to a lesser extent from phenylalanine. You can also monitor mostly tryptophan emission at the cost of a lower signal by fixing the excitation wavelength at 295 nm. The fluorescence intensity can either increase or decrease upon binding, depending on the protein. Information on how to perform calculations to obtain the binding constant for 1:1 binding can be found here: https://www.researchgate.net/publication/8065951_Receptor_fragment_approach_to_the_binding_between_CCK8_peptide_and_cholecystokinin_receptors_a_fluorescence_study_on_type_B_receptor_fragment_CCK(B)-R_(352-379)?ev=prf_pub.
Exhaustive information on fluorescence can be found in http://www.springer.com/chemistry/analytical+chemistry/book/978-0-387-31278-1.
Article Receptor fragment approach to the binding between CCK8 pepti...
Thank you very much for your inputs. I am curious about the fluoresence polarisation assay. If the intrinsic protein fluorescence intensity would not change upon ligand binding, and the fluorescence of the ligand is negligible, would it be theoretically possible to observe motility chances of the protein by autofluoresence? I see that a big concern would be the sensitivity as Irit said. Would the likelihood though increase in case the protein was small enough, while still having sufficient intrinsic fluorescence?
In principle, even though the intrinsic fluorescence remains unchanged upon binding (but this is not usually the case), you should still observe FP changes caused by protein rotation during the lifetime of the fluorophore excited state. However, the smaller is the protein the smaller will be the rotational relaxation time. This would result in signal depolarization, which would occur to the same extent on binding, if the ligand is so small as not to cause any appreciable volume change of the protein-ligand complex as compared to the protein. Take a look at http://lnbio.cnpem.br/spectroscopyandcalorimetry/files/2012/08/anisotropy.pdf.
It also depends on the scale of ligand. If the ligand is another small protein, the effect will be obvious. I am not sure if intrinsic fluorescence of protein can also be enhanced or quenched upon ions binding.
With FP you're observing changes to the rotation of the protein upon ligand binding. If your protein is very big and your ligand is very small, then the effect might not be obvious. If your ligand is big enough to have a significant effect on the protein's motility in solution, then in theory that should be measurable by FP.
If you have access to a fluorimeter and the reagents needed for the experiment, then the best way to answer your question is to just give it a try.
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