A guide to intrinsic protein fluorescence

Fluorescence spectroscopy is a very powerful tool for learning what is going on in a sample.

Published on
March 8, 2023
An abstract visualisation of the inner filter effect.
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The sources of fluorescence in proteins

Fluorescence spectroscopy is a powerful tool for learning what is happening in a sample. The minimal requirement is that there must be something in the sample that has a fluorescence signal. Fortunately for biochemists, most proteins naturally contain fluorophores: the residues of the amino acids tryptophan, tyrosine, and phenylalanine. Due to its higher extinction coefficient, tryptophan is often the main player, despite its lower abundance. Tyrosine, being more common but not as prone to absorb light, also contributes to absorbance, but its fluorescence can often be quenched by nearby tryptophan.

What fluorescence tells us about proteins

Much of the interest in protein fluorescence comes from the fact that it is highly dependent on the environment of the molecule. This is especially true for tryptophan. A tryptophan residue buried in the core of the protein will be in a less polar environment than one exposed on the surface. This difference is associated with a shift in the fluorescence maximum from shorter wavelengths in a nonpolar environment to longer wavelengths in a more polar environment.

For this reason, the fluorescence spectrum of a protein will change if the protein undergoes a change that affects the local environment of one or more of its tryptophan residues. This is very often used in studies on protein-ligand binding, protein conformational changes, or unfolding.

Fluorescence spectra of the amino acids phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). The signals have been normalized for easier viewing. Note that the details of the spectra may differ depending on the protein environment.

The advantage of studying full spectra

Changes in protein intrinsic fluorescence can be encoded by changes not only in the maximum intensity, but also in the wavelength at which this maximum occurs. This phenomenon can sometimes be difficult to observe, as it is impossible to separate the two processes when only the aggregated intensity or the intensity at a single wavelength is collected. However, by collecting the full spectrum, it is possible to obtain a much fuller and more accurate picture of the changes occurring in protein intrinsic fluorescence. This can be especially important in the study of protein-protein interactions, where even small changes in fluorescence can indicate important structural or functional differences.

Absorbance spectra of the amino acids phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). The signals have been normalized for easier viewing. Note that the details of the spectra may differ depending on the protein environment.
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