Chemical protein folding probes to quantify folded and functional cellular proteins to understand proteostasis network functions (753.1)

Abstract

While much is known about protein folding in buffers, current methodology is limited in probing how protein folding in vivo is influenced by the cellular proteostasis network quantitatively, particularly with regard to partitioning client proteins between folded and functional, soluble and misfolded, and aggregated conformational states in the face of genetic and environmental stresses. Herein we develop protein‐of‐interest folding probes that specifically recognize the functional and folded fraction of the protein‐of‐interest, , and serve to quantify the amount of folded and functional protein in cell lysate. Rapid depletion of ATP levels during cell lysis minimizes folding equilibria shifts by creating sufficient cellular chaperone holdase activity. The folding probes strategy is exemplified with retroaldolase (RA), a de novo designed enzyme, and transthyretin, a tetrameric plasma protein. This strategy revealed that partitioning of RA and TTR mutants between the aforementioned conformational states was strongly influenced by cytosolic proteostasis network perturbations, including expression of a polyglutamine protein.