Intrinsic fluorescence of proteins is a valuable property that is widely exploited for investigation of unfolding, refolding, disulfide bonding and stability studies of individual proteins and domains. It is also valuable for studying protein-protein and protein-ligand interactions including the determination of reaction/binding kinetics.
This intrinsic fluorescence is due to the presence of specific aromatic amino acids throughout the protein/peptide and their relative location within the secondary structure and local environment. That’s aromatic in the planar rings joined by covalent bonds sense, not having a lovely smell!
Fluorescence Properties of Amino Acids
Three amino acids contribute to the fluorescence of proteins: tryptophan, tyrosine and phenylalanine.
Tryptophan is the most highly fluorescent amino acid (with the highest extinction coefficient) and is the most widely studied. With an absorption maxima of 280 nm, tryptophan fluorescence is due to π to π* transitions in the indole ring. Aqueous solutions of tryptophan emit with a wavelength maximum of approximately 350 nanometers, but the intensity of emission is susceptible to quenching by water. In a protein, the emission maximum of tryptophan can vary from 307 to 353 nanometers. The wavelength, maximum and intensity of emission is highly dependent on the immediate environment around the tryptophan sidechain. Buried tryptophan residues in folded proteins exhibit a spectral shift of up to 20 nm due to their hydrophobic environment.
Tyrosine is the second most fluorescent amino acid and has an absorption maximum at around 275 nm. Although less fluorescent than tryptophan, tyrosine is often present in large numbers in many proteins. Studies based on tyrosine fluorescence are usually limited to tryptophan free proteins due to its participation in Förster resonance energy transfer. Tyrosine fluorescence can be quenched by nearby tryptophan moieties via resonance energy transfer or by ionization of its aromatic hydroxyl group.
Phenylalanine is very weakly fluorescent and can only be observed in the absence of both tryptophan and tyrosine. Due to tryptophan’s greater absorptivity, higher quantum yield, and resonance energy transfer, the fluorescence spectrum of a protein containing all three amino acids usually resembles that of tryptophan.
Absorption Wavelength (nm) Absorption coefficient (ε) (M−1cm−1) Wavelength (nm) Quantum Yield Trp (Tryptophan) 280 5600 348 0.2 Tyr (Tyrosine) 275 1420 303 0.14 Phe (Phenylalanine) 257 197 282 0.04Applications of fluorescence spectroscopy for protein analysis a governed by three principal factors; the dynamic nature of the signal, it’s localised nature and its redundancy. Fluorescence spectroscopy is a probe sensing changes in the local environment of the fluorophore which distinguishes it from generalised techniques like calorimetry and far UV CD.
Aromatic Amino Acids for Protein Concentration Determination
One of the most common practical uses for aromatic amino acid absorption is in the spectrophotometric calculation of protein concentration using the Beer-Lambert law. The Beer-Lambert law states that the absorbance of a solution is proportional to the concentration of the absorbing species and the path length of the light through the solution. Since aromatic amino acids absorb light at specific wavelengths, the number of these amino acids in a protein will contribute to the extinction coefficient (ε), which can be used to determine the concentration of a protein in solution.
A = ε c l
A: absorbance, ε: molar extinction coefficient, l: path length (cm), and c: concentration
One thing to be aware of using this technique is the presence of nucleic acids in the sample, which absorb in the 260nm range. An example of typical absorption spectra for protein and nucleic acid is shown below – for many partially purified protein samples, a shoulder appears on the peak from nucleic acid present.
Tryptophan Fluorescence for Structure and Function of Proteins
As already touched upon, the fluorescence emission of tryptophan residues is in general higher when this amino acid is on the outside of a protein and open to the environment. Fluorescence emission is therefore a key indicator of the conformational state of a protein and its folding process. Aggregation can bury surface exposed residues and cause a decrease in fluorescence, conversely unfolding can expose buried tryptophan’s and increase the signal. In complex proteins this may be a dynamic process and it is important to look at the process as well as the net effect. Understanding the fluorescence spectra of the target protein in its native state is important for looking at changes due to stability.
Tryptophan fluorescence is therefore very useful in protein unfolding studies and unfolding constants can be calculated from the time data using Michaelis-Menten equations. A typical kinetic unfolding assay would use a plate reader which adds a suitable denaturant to protein in a microplate. The fluorescence emission of the intrinsic tryptophan residues would be monitored over time. Heating can also used for thermal denaturation.
Protein-Protein and Protein-Ligand Interactions
Changes in fluorescence may be observed due to conformational changes within proteins upon binding to a substrate, ligand, chaperone or other moiety. This is possible using both intrinsic fluorescence from tryptophan, and also from engineering tryptophan residues into recombinant proteins as point mutation for monitoring purposes, while maintaining normal folding and activity.
Once example of this was study of the bacterial virulence factor immunoglobulin binding Protein L, which binds to kappa light chains found on two thirds of mammalian antibodies. Site directed mutagenesis added a tryptophan into both of the two binding sites (high affiinty and low affinity) This combined with stopped flow fluorimetry allowed the determination of binding constants.
For all protein studies, it is worth remembering that the fluorescence change observed for any interaction study may be an overall increase or decrease, depending on the conformational changes that occur within the protein(s). In some circumstances, there may even be a net zero effect in terms of intensity, due to local increases and decreases, although the wavelength may shift slightly due to these changes. An example of emission spectra series for protein binding ligand in increasing concentrations is shown below.
References and Relevant Literature
Vivian, J.T and Callis, P.R (2001) Mechanisms of Tryptophan Fluorescence Shifts in Proteins, Biophys J., 80 (5), 2093-2109.
Biter, A.B, Pollet ,J, Chen, W.H, Strych, U, Hotez, P.J and Bottazzi, M.E. (2019) A method to probe protein structure from UV absorbance spectra. Anal Biochem. 587, 11345
Graille, M., et al. (2001) Complex between Peptostreptococcus magnus Protein L and a Human Antibody Reveals Structural Convergence in the Interaction Modes of Fab Binding Proteins. Structure, 9 (8), 679 – 687.
Yammine, A., Gao, J., & Kwan, A. H. (2019). Tryptophan Fluorescence Quenching Assays for Measuring Protein-ligand Binding Affinities: Principles and a Practical Guide. Bio-protocol, 9(11), e3253.
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