His-tag based supramolecular biopolymerization

The term supramolecular polymer has been applied to polymeric materials in which the individual units, i.e., building blocks-are bound to each other via noncovalent interactions, including electrostatic or hydrogen bonding, as well as metal-ligand conjugation. The building blocks are generally low molecular weight amphiphiles. Methods for preparing biopolymers based on non-toxic, metal-ligand conjugation have been little studied; however, they offer significant potential for tuning the response of biologically relevant macromolecules. In this communication, we characterize the assembly and morphology of supramolecular biopolymers in which the building blocks are low- or medium-molecular weight globular proteins-ubiquitin and Cas9-interacting via metal-ligand conjugation. In each case, the protein gene was expressed in cell culture with the addition of hexa-His/linkers at both the N and C termini. Divalent cations investigated were Zn²⁺ and Ni²⁺. We observe in cryo-TEM imaging an absolute requirement for divalent cations for the formation of supramolecular biopolymers. In the presence of Ni²⁺, 1D assembled fibers are predominant, while with Zn²⁺, the more frequently detected structures are sheet-like. We use gel electrophoresis and CD spectroscopy to monitor possible secondary and tertiary structural changes in the protein building blocks during conjugation.