Fluorescence Spectroscopy - Tryptophan Fluorescence

Tryptophan Fluorescence

The fluorescence of a folded protein is a mixture of the fluorescence from individual aromatic residues. Most of the intrinsic fluorescence emissions of a folded protein are due to excitation of tryptophan residues, with some emissions due to tyrosine and phenylalanine; but disulfide bonds also have appreciable absorption in this wavelength range. Typically, tryptophan has a wavelength of maximum absorption of 280 nm and an emission peak that is solvatochromic, ranging from ca. 300 to 350 nm depending in the polarity of the local environment Hence, protein fluorescence may be used as a diagnostic of the conformational state of a protein. Furthermore, tryptophan fluorescence is strongly influenced by the proximity of other residues (i.e., nearby protonated groups such as Asp or Glu can cause quenching of Trp fluorescence). Also, energy transfer between tryptophan and the other fluorescent amino acids is possible, which would affect the analysis, especially in cases where the Förster acidic approach is taken. In addition, tryptophan is a relatively rare amino acid; many proteins contain only one or a few tryptophan residues. Therefore, tryptophan fluorescence can be a very sensitive measurement of the conformational state of individual tryptophan residues. The advantage compared to extrinsic probes is that the protein itself is not changed. The use of intrinsic fluorescence for the study of protein conformation is in practice limited to cases with few (or perhaps only one) tryptophan residues, since each experiences a different local environment, which gives rise to different emission spectra.

Tryptophan is an important intrinsic fluorescent probe (amino acid), which can be used to estimate the nature of microenvironment of the tryptophan. When performing experiments with denaturants, surfactants or other amphiphilic molecules, the microenvironment of the tryptophan might change. For example, if a protein containing a single tryptophan in its 'hydrophobic' core is denatured with increasing temperature, a red-shifted emission spectrum will appear. This is due to the exposure of the tryptophan to an aqueous environment as opposed to a hydrophobic protein interior. In contrast, the addition of a surfactant to a protein which contains a tryptophan which is exposed to the aqueous solvent will cause a blue-shifted emission spectrum if the tryptophan is embedded in the surfactant vesicle or micelle. Proteins that lack tryptophan may be coupled to a fluorophore.

With fluorescence excitation at 295 nm, the tryptophan emission spectrum is dominant over the weaker tyrosine and phenylalanine fluorescence.