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Team Shares Proteolysis-Based Approach to Assess Protein Folding Stability at 'Mega-Scale'

In Nature, a team from the US, Japan, France, and Israel outlines a strategy for measuring thermodynamic folding stability across 'mega-scale' protein domain sets. Using their high-throughput complementary DNA (cDNA) display proteolysis assay, the investigators report they can gauge protein folding stability patterns in a library of up to 900,000 sequences per week with a reagent price tag of around $2,000. For the study, the authors used this cDNA display proteolysis strategy to systematically come up with about 776,000 high-quality folding stability measurements for hundreds of naturally occurring or engineered protein domains containing single or double amino acid mutations under consistent conditions, for example, making it possible to consider everything from the folding stability contributions of specific amino acids to the interactions affected by protein domain folding stability-related selective pressures. "Compared with mass spectrometry-based high-throughput stability assays, cDNA display proteolysis achieves a 100-fold larger scale and can easily be applied to study mutational libraries that pose difficulties for proteomics," the authors report. "Compared with the previous yeast display proteolysis method, cDNA display proteolysis resolves a wider dynamic range of stability and is more reproducible even at a 50-fold larger experimental scale."