Enzyme-Mediated Temporal Control over the Conformational Disposition of a Condensed Protein in Macromolecular Crowded Media

Temporal regulation between input and output signals is one of the hallmarks of complex biological processes. Herein, we report that the conformational disposition of a protein in macromolecularly crowded media can be controlled with time using enzymes. First, we demonstrate the pH dependence of bovine serum albumin (BSA) condensation and conformational alteration in the presence of poly(ethylene glycol) as a crowder. However, by exploiting the strength of pH-modulatory enzymatic reactions (glucose oxidase and urease), the conversion time between the condensed and free forms can be tuned. Additionally, we demonstrate that the trapping of intermediate states with respect to the overall system at a particular α-helix or β-sheet composition and rotational mobility can be possible simply by altering the substrate concentration. Finally, we show that the intrinsic catalytic ability of BSA toward the Kemp elimination (KE) reaction is inhibited in the aggregated form but regained in the free form. In fact, the rate of KE reaction can also be actuated enzymatically in a temporal fashion, therefore demonstrating the programmability of a cascade of biochemical events in crowded media.