(484g) Signal-Responsive Structural Transitions in Asymmetric Protein Assemblies

Synthetic cells capable of sensing and responding to environmental cues provide a powerful platform for mimicking essential functions of living systems. However, achieving such stimulus-responsive behavior using protein-based materials remains challenging. Previously, we developed recombinant protein vesicles formed from amphiphilic building blocks composed of sensory domains, FRB- or FKBP-fused to fluorescent proteins and a leucine zipper (FRB-mCherry-Z_E or FKBP-sfGFP-Z_E), co-assembled with a counter leucine zipper–elastin-like polypeptide (Z_R-ELP). These vesicles exhibited inter-vesicle interactions upon rapamycin addition through the formation of FKBP–rapamycin–FRB ternary complex.

Building on this platform, we explore the formation and signal-induced structural transition of asymmetric protein assemblies under macromolecularly crowded conditions, which enhance phase separation and spatial organization. In natural systems, sensing events often trigger internal restructuring of membrane-less compartments, such as nucleoli or stress granules. We aim to replicate these post-sensing structural changes within self-assembled fusion protein materials by engineering an aqueous two-phase system (ATPS). We observe morphological rearrangements upon rapamycin addition, indicating that our protein assemblies can reorganize in response to chemical signals—mirroring stimulus-induced remodeling in natural cellular compartments. We employ fluorescence recovery after photobleaching (FRAP) to investigate these dynamics and plan to implement Förster resonance energy transfer (FRET) to assess molecular proximity and interactions. This work provides a tunable platform for studying how sensing and environmental context influence synthetic compartments' behavior.