PR.Panta Control outputs several onset parameters. In general, onsets mark temperatures at which a certain process begins, and the sample starts to change - this can either be unfolding, size increase or aggregation. The onset is useful to determine the sample handling temperatures, to stay below its onsets of unfolding, size increase or aggregation, in order to ensure that the protein is still intact.

All onsets are output by Prometheus software as ON in the data table. To differentiate between onsets of unfolding, of size increase, and of aggregation in the software, look at the data it was generated from (ratio for unfolding, cumulant radius for size increase, and scattering intensity and turbidity for aggregation).

Be aware that scattering data is only available if data was generated with measurement types supporting DLS and with Dynamic Light Scattering set to on.

Onset of unfolding

The onset of unfolding has many names: onset of melting, Ton, Tonset. In Prometheus software, it is output simply as ON or as ON (Ratio) to indicate that it was derived from ratio data.

The onset of unfolding can be a useful parameter. It is typically regarded as the temperature to keep your sample below. It can also help when comparing samples with similar inflection points (IP) or melting points (Tm) as illustrated in the figure below. In this example, the unfolding profile of two proteins is shown. Although the two proteins exhibit very similar IPs/Tms, their onsets of unfolding clearly differ. The protein on the left starts to unfold at a much higher temperature, meaning this protein will be more stable at most temperatures below the IP/Tm.

In addition to the fluorescence ratio data, the first derivative is also shown in the lower panels for better visualization.

Onset of size increase

The onset of size increase is called Tsize and indicates a change in the average particle size (the cumulant radius) calculated from dynamic light scattering data. In PR.Panta Control it is output as ON or ON (Radius).

Just like the onset of unfolding, Tsize is typically regarded as a temperature to keep your sample below. It indicates a major conformational or structural change to the particles in solution, which translates to several possible events on a molecular level: cooperative unfolding, oligomerization or formation of the first aggregates. Which of these events underlie the observed size change is best analyzed by comparing the cumulant radius data to the corresponding unfolding and/or aggregation profiles. For proteins showing more than one unfolding event, size increase may happen as a consequence of the first unfolding event or of a later one. In contrast to the scattering intensity or turbidity signals, the cumulant radius is also sensitive towards smaller structural changes, which is why Tsize often precedes the onsets of aggregation. Please note that no onset is output for the size distribution profile, but the data may help to identify the underlying aggregation pathway (see figure below).

Onset of aggregation

PR.Panta Control outputs two onsets of aggregation: The onset of scattering, Tscattering, from scattering data, and the onset of turbidity, Tturbidity from turbidity data. In Prometheus software, they are displayed as ON or ON (Scattering) and ON (Turbidity), respectively.

Just like the other onsets, the onsets of aggregation are typically regarded as a temperature to keep your sample below, as aggregation is unwanted in most experimental settings. The onset of aggregation correlates with the unfolding of the protein. For proteins showing more than one unfolding event, aggregation may happen as a consequence of the first unfolding event or of a later one. While the onset of turbidity marks heavy aggregation and precipitation as well as formation of particles larger than 25 nm in diameter, the onset of scattering is sensitive to minute amounts of aggregates, very small aggregates, and low concentrations. Depending on the type and rate of aggregation, the two onsets may or may not correlate.

Figure: Unfolding and aggregation profiles of a protein in two different buffers (red/blue), showing the fluorescence ratio 350/330 nm, the cumulant radius, the size distribution, and the turbidity as a function of temperature. The onset of unfolding (Ton), the inflection points (IP), the onset of size increase (Tsize) and the onset of aggregation (Tturbidity) are shown in blue/red for each sample. The scattering intensity data and Tscattering are not shown, because this parameter was similar to Tsize in this dataset. In the example in red, Tsize coincides with the first unfolding transition, while Tturbidity coincides with the second transition. Comparing all profiles to the size distribution profile aids the interpretation: Unfolding of the first domain results in conformational changes and cooperative unfolding, while unfolding of the second domain promotes heavy aggregation and precipitation (visible as speckle pattern). In the example in blue, unfolding of the first domain results in no apparent size increase, while upon unfolding of the second domain particle sizes increases (Tsize). The size distribution profile shows that aggregation/oligomerization happens in an ordered fashion. The formed oligomers are too small to be detected with the backreflection optics and thus no Tturbidity is determined.