Title: Revealing kinetics of protein unfolding with a high-throughput microfluidic platform

Abstract: Proper protein folding is essential for a linear polypeptide chain to form a functional three-dimensional structure. While thermodynamic stability determines the fraction of protein molecules that are folded at equilibrium, kinetic stability has been proposed to play a crucial role in regulating proteins by tuning their abundance and lifetime in the cell. Currently, we cannot accurately predict mutational effects on protein unfolding rates, which is critical for identifying potential disease-associated mutations, manipulating proteins for therapeutic purposes, and designing proteins with novel functions. To reach this level of understanding, we need tools to make quantitative kinetic stability measurements at a scale that approaches the vast number of possible sequences. To address this, we developed SPARKfold (Simultaneous Proteolysis Assay Revealing Kinetics of Folding), a microfluidic platform that enables expression, purification, and measurement of unfolding rates for 1104 protein variants in parallel. SPARKfold enables quantitative analysis of protein kinetic stability at an unprecedented scale, with significant time and cost savings compared to current approaches. To demonstrate the power and potential of SPARKfold, we applied it to determine unfolding rates for a library of dihydrofolate reductase (DHFR) orthologs and variants, revealing how sequence, structure, and conservation contribute to kinetic stability. We found that single mutations in DHFR increase unfolding rates by up to 30-fold, establishing that many mutations destabilize proteins by increasing the unfolding rate. By comparing mutational effects on both thermodynamic and kinetic stability, we provide novel insights into the folding pathway of DHFR. In the future, SPARKfold will enable rapid characterization of variants across protein systems, revealing how mutations drive misfolding and aggregation in disease and informing the development of therapeutics. More broadly, SPARKfold will reveal biophysical principles governing kinetic stability, providing a basis to study and engineer protein folding.

Please contact Madelyn Bernstein for the Zoom link