Noncanonical\nAmino Acid Incorporation Modulates Condensate\nStates of Intrinsically Disordered Proteins in <i>Escherichia\ncoli</i> Cells

Biomolecular condensates are distinct\nsubcellular structures\nwith\non-demand material states and dynamics in living cells. However, strategies\nfor modulating their material states and physicochemical properties\nare lacking. Here, we report a chemical strategy for modulating the\ncondensate states of intrinsically disordered proteins in bacterial Escherichia coli cells. This is achieved by noncanonical\namino acid (DOPA) incorporation into model resilin-like proteins (RLPs)\nto endow autonomous oxidative and coordinative cross-linking mechanisms.\nBiogenesis of spherical gel-like condensates is achieved upon heterologous\nexpression of the DOPA-incorporated RLP in the cells at 30 °C.\nWe reveal that liquid–liquid phase separation underlies the\nformation of liquid condensates, and this liquid-like state is metastable\nand its dynamics is compromised by the oxidative and coordinative\ncross-linkings that ultimately drive the liquid-to-gel transition.\nTherefore, this study has deepened our understanding of biomolecular\ncondensation and offers a new chemical strategy to expand the landscape\nof condensation phenotypes of living cells.