The stability of proteins is crucial in many in vitro protein studies and is considered a major requirement in functional studies involving native and recombinant proteins. Thus, understanding protein stability and preserving the native conformation and normal functions of your protein of interest can be very helpful when working with your protein of interest.
However, it is important to note that proteins are in a state of constant flux and have specifically defined half-lives. They are constantly being synthesized and degraded inside the cell to reduce unnecessary protein load, facilitate the removal of proteins that have done their jobs, and to prevent any undesirable effects arising from the constitutive action of certain proteins.
Additionally, their structural and functional properties can easily be affected by unfavorable conditions or sudden variations in their native environment. An abrupt change in temperature or pH, the presence of proteases, heavy metal ions, cellular debris and other precipitated materials, the absence of the right additives (e.g., ethylene glycol, glycerol, cryoprotectants, antimicrobial agents, etc.) and/or mechanical agitation can affect the stability of the protein and subsequently alter its properties and functions. You don’t want this to happen since it can lead to degradation, denaturation or precipitation and render your protein useless.
Determining Protein Stability: Some of the Most Common Methods Used
Over the years, a number of experimental methods have been used to determine and measure protein structure and stability. Here are some of them.
Differential Scanning Calorimetry (DSC)
This technique has been widely used in characterizing the stability of proteins in their native form by measuring the amount of heat required to denature a particular biomolecule (e.g., protein). Generally, molecules with higher thermal transition midpoint (Tm) are considered more stable than those with lower transition midpoints.
Due to its accuracy and high reproducibility, DSC is recognized as the “gold standard” for thermal stability analysis and can be used in characterizing and selecting the most suitable proteins in biotherapeutic development as well as for ligand interaction studies.
Additionally, DSC offers the following advantages over other protein stability determination methods:
- Simple sample preparation
- Thermodynamic parameter determination
- Can use solid and liquid samples
- Can be used to detect denaturation temperatures and irreversible-reversible phase changes
Pulse-Chase Method
Despite the fact that it involves labeling cells with radioactive precursors or pulse, the Pulse-Chase method has traditionally been the method of choice for determining protein stability. Many researchers favor this method since it allows for the accurate measurement of protein half-life and the determination of its subcellular localization.
There are at least two popular non-radioactive versions of this method – the bleach-chase method and the cycloheximide chase method.
Bleach-chase method. In this method, the protein of interest is fused with a fluorophore and bleached with a brief pulse of light to produce a fluorescently and non-fluorescently protein population. The rate of degradation is correlated with the rate of fluorescence recovery after the bleaching process.
Cycloheximide-chase method. Instead of adding radioactive precursors, cycloheximide is added to inhibit protein synthesis and allow researchers to observe and assess the degradation of proteins as a function of time.
Circular Dichroism (CD) Spectroscopy
CD spectroscopy provides information on the conformation and stability of proteins by measuring the differences in absorption between left- and right-handed polarized light resulting from the structural asymmetry of the protein of interest. While it provides low-resolution information, CD spectroscopy is commonly used in most laboratories because it only requires a small amount of sample, is non-destructive and relatively easy to operate.
Fluorescence-based Activity Assays
Basically, fluorescence-based activity assays can be used to indirectly measure protein stability and provide information on protein activity and functionality. However, since it is highly susceptible to artifacts, coming up with reproducible results can be a challenge. In addition, the dyes used to track the stability of the proteins can affect the stability of the protein of interest.
