Unfolding profiles can have very different shapes, depending on the composition and position of the protein's fluorescent amino acid residues.

In the folded state, Tryptophan residues (Trp) are often buried in the hydrophobic core of a protein, which leads to the fluorescence emission peaking around 330 nm. They then become surface-exposed during unfolding, which often shifts the fluorescence emission peak toward 350 nm. Since this is a shift towards the red end of the light spectrum, the result is often called a redshift. The resulting S-shaped ratio unfolding profile is the most common (top row in the figure below). Sometimes, the unfolding profiles look reversed, called a blueshift (second row in the figure below).

In proteins where Trp residues predominate but are already surface-exposed in the native state, the fluorescence emission maximum for the native protein is often closer to 350 nm than to 330 nm. The unfolding process then may not trigger a shift, just a change in fluorescence intensity, since the environment of the Trp residues does not change as much upon unfolding. Often, the fluorescence at 350 nm exhibits a higher degree of quenching by temperature, meaning that the fluorescence decrease is larger at 350 nm than at 330 nm (first example in the second row).

In proteins that contain many Tyrosine (Tyr) residues but few Trp, the Tyr signal can dominate. Tyrosine residues may exhibit a small shift in the emission maximum peak towards the blue end of the spectrum, or simply show a change in emission intensity, especially around the 350 nm window. This can also lead to a reverse-S-shaped profile (second example in the second row).

Some proteins also show no shift upon unfolding but show a similar change in emission intensity for both 330 nm and 350 nm. In such cases, the unfolding process is still visible in single wavelength view but may not be visible in the ratio view (bottom row of the figure below). If a protein unexpectedly shows no transition in the ratio view, it is always recommended to check the single wavelength views.

Figure: The left column shows exemplary fluorescence emission spectra for folded proteins (solid lines) and increasingly unfolded proteins (dotted lines). Blue and purple lines highlight the wavelengths at which Prometheus detects fluorescence (blue line for 330 nm, purple line for 350 nm). The next column shows the resulting fluorescence intensity measured over temperature. Fluorescence generally decreases with increasing temperature. In addition to this general decrease, characteristic inflections can be seen as the protein unfolds. The blue line shows the fluorescence signal at 330 nm, the purple line shows the signal at 350 nm. The right column shows the ratio signal over temperature, derived from the single wavelength signals.