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Thermal Stability Assessment

by Thermal Ramp Experiments

Whether in academia or industry, whether proteins or nucleic acids—monitoring temperature-dependent circular dichroism is the most direct structure-based approach to evaluating thermal stability.

Thermal Ramping Intro Graphs 1

Obtain thermodynamic parameters

By following, e.g., protein unfolding upon heating by monitoring the spectroscopic signal at a particular wavelength (single-temperature ramps), a sigmoidal unfolding curve is obtained.

This unfolding curve is characterised by an inflection point where half of the protein molecules are still folded whereas the other half is unfolded—this melting temperature, Tm, is a measure of thermal stability: the higher the Tm, the higher the stability.

Moreover, such temperature ramp experiments also yield the van't Hoff enthalpy, ΔHvH, which is related to the width of the unfolding transition—the sharper the transition, the larger ΔHvH.

Thermal Ramping Intro Graphs 2

Observe structural changes

By measuring full spectra at multiple, constant temperatures (stepped temperature ramps), you can see how elevated temperatures affect the structure of your molecule.

Thermal Ramping Intro Graphs 3

More information in the same time

Classical approaches as above only give you part of the picture:

  • Single-wavelength temperature ramps yield data at high temperature resolution, but provide no structural information.
  • Stepped temperature ramps provide structural information, but yield data at a poor temperature resolution.

The advantages of both approaches can be combined in continuous, multiwavelength temperature ramps, where full spectra are measured upon continuous heating of the sample.

This implies that the temperature changes during acquisition, and this is accounted for with the right analysis software, which globally fits all unfolding curves to yield precise thermodynamics from structural information at high temperature resolution.



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