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.