A Cofactor-Activated Molecular Switch for Condensate Biogenesis and Catalysis in Escherichia coli

Abstract

Biomolecular condensates formed through liquid-liquid phase separation (LLPS) compartmentalize biochemical reactions without enclosing membranes, enabling spatiotemporal control over diverse cellular processes. Engineering genetically encoded proteins that phase separate in response to defined chemical inputs remains a central challenge for synthetic biology. Here, we report a coiled-coil peptide polymer, M1, that undergoes cofactor-dependent condensation both in vitro and in Escherichia coli. M1 is an ABA triblock construct comprising two terminal helical domains connected by a flexible, intrinsically disordered linker. The terminal domains are derived from a heme-responsive coiled-coil motif that is destabilized in the apo state but assembles into a four-helix bundle upon metalloporphyrin coordination. We demonstrate that M1 forms condensates exclusively in its cofactor-bound state, both in vitro and in cells. In E. coli, these intracellular condensates accumulate at the cell poles in a concentration-dependent manner. Depletion of cellular heme biosynthetic capacity suppressed condensate formation, which was rescued by supplementation with the heme precursor {delta}-aminolevulinic acid ({delta}-ALA) and iron, consistent with metalloporphyrin coordination triggering assembly. The condensates retain peroxidase activity characteristic of heme-containing proteins and catalyze the oxidation of Amplex Red to resorufin both in vitro and in living cells. These results establish metalloporphyrin binding as a molecular switch for condensate biogenesis in a structured peptide polymer, directly coupling cofactor coordination, mesoscale assembly, and catalytic function within a single designed system. SYNOPSIS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/734084v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1bc678eorg.highwire.dtl.DTLVardef@32bb4corg.highwire.dtl.DTLVardef@a6890aorg.highwire.dtl.DTLVardef@16f13c2_HPS_FORMAT_FIGEXP M_FIG C_FIG