Stability Analysis

Thermal Shift Assays

Source - Instruct

Source – Instruct

Protein stability can be determined based on the time it takes for protein structure to denature in heat (also known as reaching the ‘melting temperature’). Comparison of melting temperature, and its shift due to structural changes in proteins, can be useful in determining the stability of the protein. A common way to accomplish this is by use of a fluorescent dye that binds the hydrophobic residues in the protein.

Typically, in a folded protein, these hydrophobic residues move to the interior of the protein, as their hydrophobicity drives folding by moving these residues away from the aqueous environment. Therefore, the melting temperature is measured by mixing a sample of protein with the aforementioned fluorescent dye, and heating the sample. As the protein melts and loses structure, the interior hydrophobic residues are exposed, binding to the dye and generating observable fluorescence. This can be measured using typical biological lab equipment such as a plate reader for high-throughput screening experiments. Measurement of the thermal unfolding curve allows calculation of t1/2, the half life time of the protein, a measure of protein stability. Some common uses of this measurement include comparing wild-type and modified proteins, as well as analyzing the aggregation state of a protein.

Circular Dichroism

Source - Wikipedia

Source – Wikipedia

Another measurement for protein stability is circular dichroism, the measurement of the change of ellipticity of light due to the chirality of molecules. This is a common technique for measuring the secondary structure of proteins. The circular dichroism spectrum of a dilute protein solution is measured and compared with a database of proteins with known structure. The spectrum is fit using an algorithm to compare it to the known structures, and percentages of alpha helices, beta sheets, etc are generated.

This technique allows comparison of folding between wild-type and modified proteins, and the folded state of protein can be evaluated after it has been processed. It also permits study of the effect of pH, temperature, and other environmental alterations on protein folding.

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