Genetic Fusion of Thermoresponsive Polypeptides with UCST-type Behavior Mediates 1D Assembly of Coiled-Coil Bundlemers

Thermoresponsive resilin-like polypeptides (RLPs) of various lengths were genetically fused to two different computationally designed coiled coil-forming peptides with distinct thermal stability, to develop new strategies to assemble coiled coil peptides via temperature-triggered phase separation of the RLP units. Their successful production in bacterial expression hosts was verified via gel electrophoresis, mass spectrometry, and amino acid analysis. Circular dichroism (CD) spectroscopy, ultraviolet-visible (UV/Vis) turbidimetry, and dynamic light scattering (DLS) measurements confirmed the stability of the coiled coils and showed that the thermosensitive phase behavior of the RLPs was preserved in the genetically fused hybrid polypeptides. Cryogenic-transmission electron microscopy and coarse-grained modeling revealed that functionalizing the coiled coils with thermoresponsive RLPs leads to their thermally triggered noncovalent assembly into nanofibrillar assemblies.

Automated summary

Researchers genetically fused thermoresponsive resilin-like polypeptides (RLPs) of various lengths with coiled coil-forming peptides to develop new strategies for assembling coiled coil peptides via temperature-triggered phase separation. Their successful production was confirmed through various analyses. The stability of the coiled coils and the thermosensitive phase behavior of the RLPs were preserved in the genetically fused hybrid polypeptides, leading to their noncovalent assembly into nanofibrillar structures when triggered by temperature.