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

Abstract

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 coarsegrained modeling revealed that functionalizing the coiled coils with thermoresponsive RLPs leads to their thermally triggered noncovalent assembly into nanofibrillar assemblies.

Encoding Structure in Intrinsically Disordered Protein Biomaterials

“…They demonstrated that the coacervation of the RLPs upon cooling below their UCST combined with the propensity of the rigid, orderpromoting peptides to remain intact enables control over nanofibrillar (1D) self-assembly. 33 Stimuli-responsive triblock copolypeptides composed of ELPs and ligand-responsive calmodulin (CalM) having helical structure have also been recombinantly synthesized. 34 Upon heating dilute solutions of ELP-CalM-ELP above the T t , these triblock copolypeptides self-assemble into nanoscale vesicles with diameters of ∼240−455 nm, depending on salt-and ligand-induced conformational changes of the middle CalM blocks.…”

“…The Kiick group also functionalized RLPs with computationally designed coiled-coil-forming peptides. They demonstrated that the coacervation of the RLPs upon cooling below their UCST combined with the propensity of the rigid, order-promoting peptides to remain intact enables control over nanofibrillar (1D) self-assembly …”