Subtle changes in a protein’s amino acid sequence can have profound effects on a protein’s energy landscape. Even mutations that preserve a protein’s native fold may make high-energy pathogenic or proteolytically susceptible states more accessible. However, we lack a predictive understanding of the relationship between protein sequence, dynamics, and stability. Large data sets linking changes in protein sequence with perturbations in protein energetics may allow the field to approach these questions from new angles. Therefore, we have developed a high throughput assay coupling quantitative click chemistry with a yeast display system to measure the effect of sequence changes on protein energetics and dynamics. We can incorporate a click chemistry probe into a buried site on a protein and monitor the rate of modification of this probe. Depending on the kinetic regime in which this modification occurs, this observed rate can either report on the protein’s stability or unfolding rate. Using this assay, we have identified sites at which click chemistry labeling reports on global unfolding, partial unfolding, and misfolding. In addition to expanding our basic knowledge of protein folding, exploration of the relationship between sequence, kinetic barrier heights, and thermodynamic stability may contribute to a greater understanding of how novel or pathogenic protein states arise.
Gerber et al. (2026) studied this question.