If you want to gain insight into your protein’s structure and function, start by monitoring its stability. Even slight changes to your protein’s architecture or environment can significantly alter its biological activity — for better or for worse.
There are lots of questions about stability that help you understand your protein’s behavior. For example: 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? How will antibody formulations behave at higher concentrations?
With answers to these questions, you can better understand your biologic-based treatments and diagnostics.
These stability parameters will help you address questions about the biophysical characteristics of your protein preparations.
1. 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. Tm is among the most common of parameters to monitor when determining protein stability.
2. Tonset or Ton is the temperature at which a protein begins to unfold. In temperature-sensitive situations, it’s important to understand when a protein will denature and lose its activity.
3. Onset of turbidity (Tturb) is the point at which proteins exhibit a tendency to aggregate into large, amorphous particles. It’s important to measure this parameter because misfolded proteins lead to aggregation. Aggregated proteins lose their effectiveness and are associated with several disorders.
4. The Hydrodynamic radius (rH) is the size of particles in a solvated state. Knowing the size of your protein is valuable when making changes to the protein environment; slight unfolding or binding of other proteins will change the rH value.
5. The Polydispersity index (PDI) represents the distribution of size populations of your protein. A lower PDI indicates a preparation with a single-species of well-folded proteins. As the PDI increases, it’s indicative of misfolding or contaminants in your solution.
6. The second virial coefficient, B22, is a parameter that quantifies protein-protein interactions in dilute solution. It’s used to predict whether or not proteins will self-associate and therefore be more prone to aggregation at higher concentrations.
Each of these parameters provides valuable information about protein characterization. Insights from protein stability analyses improve understanding and outcomes for protein activity, binding partners, kinetics, drug development, and more.