Biomolecular condensates formed by liquid–liquid phase separation provide cells with a powerful means to spatially organize molecules and regulate biochemical activity in membraneless organelles. While most known condensates rely on intrinsically disordered proteins, RNA, or metabolites such as ATP, far less is understood about how structured proteins and cofactors can be used to control condensation in a programmable manner. Here, we describe two complementary systems that demonstrate how metal cofactors can be used as molecular switches to couple protein folding, multivalency, and phase separation, thereby controlling both spatial organization and chemical function.
In the first system, a designed coiled-coil peptide polymer underwent phase separation only upon heme coordination. Cofactor binding induces folding and higher-order assembly, resulting in condensate formation both in vitro and in Escherichia coli. Condensation is governed by concentration thresholds, metal availability, and electrostatic interactions with the bacterial nucleoid, highlighting how coordination chemistry regulates intracellular phase behavior.
The same principles were extended to construct carboxysome-inspired catalytic coacervates. Electrostatic complexation between a supercharged metalloprotein scaffold and a polypeptide drives the formation of a condensed phase that concentrates a cobalt porphyrin catalyst, photosensitizer, electron donor, and accessory enzymes. Confinement within coacervates enhances light-driven hydrogen evolution and CO₂ reduction by promoting colocalization, stabilizing reactive intermediates, and modulating the local reaction environment. The incorporation of carbonic anhydrase selectively amplifies CO₂ reduction, recapitulating key aspects of enzymatic synergy in natural carboxysomes. This study establishes metal coordination and cofactor binding as powerful chemical handles for programming biomolecular phase separation and reactivity, providing a general framework for designing functional stimuli-responsive condensates.
