His-tag based supramolecular biopolymerization

Abstract

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.

Fig. 1

Cartoon of hexa-His ([His₆]) ligand–metal conjugation with the potential of promoting self-assembly of protein fibers or sheets as macromolecular biopolymers. [His₆]-tags are added to expression genes in E. coli BL21(DE3) competent cells at both the N- and C-terminal ends of the proteins. Dimensions are not to scale.

Fig. 2

(A) Cryo-TEM imaging of fibrous protein in vitreous ice obtained approx. 10 min after addition of 10 µM NiCl₂ to 0.05 mg/mL UB-[His₆]₂ in 30 mM Tris, pH 7.5, 25 °C. (B) Cryo-TEM image of 0.05 mg/mL UB-[His₆]₂ following 10 min incubation in 30 mM Tris pH 7.5 at 25 °C with the addition of 10 mM ZnCl₂. (C) Cryo-TEM image of 3.8 mg/mL UB-[His₆]₂ following overnight incubation in 30 mM Tris pH 7.5 at 19 °C with the addition of 100 mM ZnCl₂. Folded sheets/membranes are observed. (D) Cryo-TEM image of 3.8 mg/mL UB-[His₆]₂ in in the absence of Ni²⁺ or Zn²⁺ with overnight incubation at 19 °C.

Fig. 3

(A, B) Cryo-TEM imaging of fibrous protein in vitreous ice obtained approx. 10 min after addition of 10 µM NiCl₂ to 0.05 mg/mL Cas9-[His₆]₂ in 30 mM Tris, pH 7.5, 25 °C. (C) Cryo-TEM image of 0.05 mg/mL Cas9-[His₆]₂ following 10 min incubation in 30 mM Tris pH 7.5 at 25 °C with the addition of 10 mM ZnCl₂. Folded sheets/membranes are observed. (D) Cryo-TEM image of concentrated (3.8 mg/mL) Cas9-[His₆]₂ in the absence of Ni²⁺ or Zn²⁺, incubated overnight in 30 mM Tris pH 7.5 at 19 °C. Organized supramolecular structures are not observed, rather disorganized aggregates.

Fig. 4

Far UV circular dichroism (CD) spectra of 0.05 mg/mL doubly His-tagged UB (top row A, B) or Cas9 (bottom row C, D) with 10 µM NiCl₂ (left hand panels - -) or ZnCl₂ (right hand panels - -) in DDW at 25 °C. Spectra measured in the absence of metal ions (---) were used as control. Background has been subtracted.

Fig. 5

Gel electrophoresis of [UB-His₆]₂ and [Cas9-His₆]₂: (A) SDS-PAGE with reducing agent; (B) Native-PAGE. Red arrows indicate protein bands. Gels are Coomassie stained.