A One-Pot Analysis Approach to Simplify Measurements of Protein Stability and Folding Kinetics

Publication date: Available online 19 December 2018Source: Biochimica et Biophysica Acta (BBA) - Proteins and ProteomicsAuthor(s): Yi-Kai Liu, Huei-Yu Chen, Pin Ju Chueh, Pei-Fen LiuAbstractTo achieve a good understanding of the characteristics of a protein, it is important to study its stability and folding kinetics. Investigations of protein stability have been recently applied to drug-target identification, drug screening, and proteomic studies. The efficiency of the experiments performed to study protein stability and folding kinetics is now a crucial factor that needs to be optimized for these potential applications. However, the standard procedures used to carry out these experiments are usually complicated and time consuming. Large number of measurements is the bottleneck that limits the application of protein folding to large-scale experiments. To overcome this limitation, we developed a method denoted as “one-pot analysis” which is based on taking a single measurement from a mixture of samples rather than from every sample. We combined one-pot analysis with pulse proteolysis to determine the effects of the binding of maltose to maltose-binding protein on the protein folding properties. After carrying out a simple optimization, we demonstrated that protein stability or unfolding kinetics could be measured accurately with just one detection measurement. We then further applied the optimized conditions to cellular thermal shift assay (CETSA). Combining one-pot analy...
Source: Biochimica et Biophysica Acta (BBA) Proteins and Proteomics - Category: Biochemistry Source Type: research