Over the course of the measurement, the fluorescence intensity of each sample is recorded at 330 nm and 350 nm. The recorded data can be displayed and analyzed in different ways.

In the single wavelength detection (330 nm or 350 nm), we look at the change in fluorescence intensity at a defined wavelength over temperature. The ratio 350 nm / 330 nm however is a measure for a spectral shift in the fluorescence emission profile of the Tryptophan (Trp) residues. This shift occurs in most unfolding events and is caused by the environmental change that the Trp residues undergo upon unfolding of the protein. In the folded state, Trp residues are often buried in the hydrophobic core of a protein, which leads to the fluorescence emission peaking around 330 nm. Trp residues then become surface-exposed during unfolding, which often shifts the fluorescence emission peak toward 350 nm. Since the ratio 350 nm / 330 nm typically negates effects of autofluorescent additives and general fluorescence decay with increasing temperature, this detection mode is usually more robust than single wavelength detection. Therefore, clear inflection points (and Tms) can be derived which might not be visible in the single wavelength detection modes. Alternatively, it is also possible that unfolding events which do not trigger an emission peak shift are visible in the single wavelength data, but not in the ratio. It is therefore recommended to inspect all collected data, as important structural information might be obtained by comparing the unfolding profiles in the three analysis modes.

In a case where you examine both single wavelength and ratio data for a protein and observe somewhat different IP values, there are two possible explanations on why this may happen:

• The IPs/melting points obtained from the different analysis modes deviate slightly (typically 1-2 °C): As the fluorescence of Trp and Tyrosine (Tyr) exponentially decreases with temperature, we calculate the ratio between the two wavelengths to correct for baseline drifts. This mathematical operation tilts the resulting curve as compared to the single wavelengths, resulting in a slightly deviating inflection point. This procedure however yields most reproducible data and provides further insight into structural details by revealing spectral shifts of fluorescence emission. In such a case, the melting point obtained by the 350 nm/330 nm ratio is preferably used.
• The IPs/melting points obtained from the different analysis modes deviate more than 2 °C: In this case it is likely that the two melting points reflect two distinct events (e.g. unfolding of two different domains). If Trp residues are already surface-exposed in the native state, an unfolding event might not necessarily result in change of emission wavelength (typically a redshift). This can cause a visible inflection in the single wavelength analysis but not in the ratio analysis. The melting point you see in the ratio analysis is likely the result of the unfolding of a different part of the protein structure (e.g. a different domain) where Trp residues are buried in the hydrophobic core of the protein and show a redshift of their emission spectra upon unfolding.