Tuning and Characterizing the Structure of Peptide Core-Shell Multiphasic Condensates by the Blue Fluorescent Amino...

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) play crucial roles in cellular organization and offer a versatile platform for designing synthetic organelles. Recently, extremely short peptides have been shown to assemble into highly organized core-shell multiphasic condensates, where tryptophan (Trp) residues play an indispensable role (Zhou, L.; et al.J. Am. Chem. Soc.2025, 147, 31290-31299.). Here, we rationally reprogram the architecture of these short-peptide condensates by substituting native Trp with the unnatural amino acid 4-cyanotryptophan (4CN-Trp). Using the intrinsic fluorescence and infrared reporting capabilities of 4CN-Trp, we monitor the LLPS behavior of a targeted library of 4CN-Trp-substituted peptides. We reveal that a single atomic-level substitution from Trp to 4CN-Trp fundamentally inverts the condensate architecture. In contrast to the solid-core/liquid-shell droplets formed by native Trp-containing peptides, the 4CN-Trp-containing peptides assemble into core-shell condensates featuring a dynamic, highly hydrated, liquid-like inner core enveloped by a dense, solid-like outer shell. Our findings highlight how subtle modifications to aromatic residues can dramatically modulate the macroscopic physical properties of multiphasic condensates, offering a new strategy for the precise engineering of peptide-based synthetic organelles and smart biomaterials.