To understand a given protein’s stability is to gain insight into its structure and its function. Light changes to protein architecture or its environment can significantly alter its biological activity — for better or for worse.
Does the protein unfold at a given pH? Does the presence of certain molecules cause aggregation? What changes to secondary structures cause measurable changes in activity? How can we explain its binding mechanisms? Does a recombinant protein denature before it reaches its target? These are the types of questions that are addressed by observing protein stability.
Over the years, scientists have agreed on a handful of established parameters for measuring stability. Knowing these variables enables researchers to track the tendencies of proteins to unfold or aggregate in response to temperature or chemicals.
These are the most common parameters for measuring protein stability
- The unfolding transition temperature (Tm), is the point at which 50% of the protein is unfolded. Proteins with a higher Tm are more stable because a greater input of energy is required to reach the unfolding transition. Because Tm is an accurate and established metric for assessing protein stability, it’s an essential parameter to determine.
- Tonset is the detectable temperature at which a protein begins to unfold. Particularly in temperature-sensitive situations, it’s important to understand when a protein will denature and lose its activity.
- Aggregation temperature (Tagg) is the point in which proteins exhibit a tendency to aggregate. It’s important to measure this parameter because aggregated proteins are caused by misfolding. Aggregated proteins lose their effectiveness and are also associated with several disorders.
- The Gibbs free energy (ΔG) of protein unfolding is a thermodynamic measure of the likelihood a folding event may occur. For a given event, proteins with more negative ΔG are more likely to fold. ΔG also relates protein folding to changes in enthalpy and temperature.
- Activation energy (Ea) is the amount of energy needed for a protein to unfold. This can be used as a measure of stability, in which a higher Ea indicates higher stability.
- The concentration of a denaturant that causes 50% of proteins to unfold (Cm or C50) addresses the relationship between chemical denaturants and proteins. This parameter is useful when studying chemical effects, or when protein stability is tested using denaturants.
Altogether, these parameters provide valuable information in basic protein characterization to both basic research and industry applications. Insights from protein stability can lead to new findings in protein activity, binding partners, kinetics, drug development, and more.
Learn how Prometheus measures these parameters to precisely characterize protein stability.