Genetic Fusion of Thermoresponsive Polypeptides Mediates 1D Assembly of Coiled Coil Bundlemers

Design of stimuli-responsive nanofibrillar assemblies is of interest for generating controllable nanomaterial motion for actuation in simple machines. Protein-engineering approaches provide excellent opportunities for the design and production of extremely well-defined biomimetic materials capable of organizing different types of molecular building blocks at multiple size scales. In this study, we genetically fused thermoresponsive resilin-like polypeptides (RLPs) of various lengths to two differnt computationally designed coiled coil-forming peptides with distinct thermal stability, to develop strategies to control the assembly of the coiled coil peptides on the basis of the temperature-triggered phase separation of the RLP units. This library of RLP-functionalized coiled coils was recombinantly produced in bacterial expression hosts and purified via immobilized metal affinity chromatography. Their composition and purity were confirmed via gel electrophoresis, mass spectrometry, and amino acid analysis. Ultraviolet-Visible turbidimetry measurements at 350 nm were used to validate that the thermosensitive phase behavior of the RLPs was preserved in the genetically fused hybrid polypeptides. An increase in scattering intensity upon cooling below the transition temperature was indicative of an onset of assembly. Circular dichroism spectra showed strong helicity characteristic of the coiled coil state remaining stable in the hybrid polypeptides below their transition temperature. Cryogenic-transmission electron microscopy revealed that genetic fusion of RLPs to both coiled coil-forming peptides resulted in nanofibrillar assembly of coiled coil peptide bundlemers. Our study provides opportunities for the fabrication of thermoresponsive 1D assemblies which can be used as fundamental subunits in simple molecular machines.