Green fluorescent protein nanopolygons as monodisperse supramolecular assemblies of functional proteins with defined valency

Abstract

Supramolecular protein assemblies offer novel nanoscale architectures with molecular precision and unparalleled functional diversity. A key challenge, however, is to create precise nano-assemblies of functional proteins with both defined structures and a controlled number of protein-building blocks. Here we report a series of supramolecular green fluorescent protein oligomers that are assembled in precise polygonal geometries and prepared in a monodisperse population. Green fluorescent protein is engineered to be self-assembled in cells into oligomeric assemblies that are natively separated in a single-protein resolution by surface charge manipulation, affording monodisperse protein (nano)polygons from dimer to decamer. Several functional proteins are multivalently displayed on the oligomers with controlled orientations. Spatial arrangements of protein oligomers and displayed functional proteins are directly visualized by a transmission electron microscope. By employing our functional protein assemblies, we provide experimental insight into multivalent protein-protein interactions and tools to manipulate receptor clustering on live cell surfaces.

Figure 1. Cellular self-assembly of GFP oligomers.

(a) Schematic representation of the fabrication of discrete GFP (nano)polygons. The β strand 11 of GFP is connected to the N terminus of GFP 1–10 using a short peptide linker, and the resulting GFP monomer unit undergoes self-assembly into polygonal structures in the cell. Through the introduction of supercharges on the polygon surface, GFP polygon mixtures can be separated and isolated depending on the number of GFP-building blocks, to produce a series of discrete GFP polygons. (b) SDS–PAGE analysis of GFP oligomers. Oligomer mixtures were applied to a PAGE gel containing 0.1% SDS without (lane 1) or with (lane 2) boiling. The gel was analysed by a fluorescent image analyser with 470-nm excitation and 530-nm emission filters (left), and Coomassie blue staining (right). (c) SEC of GFP oligomers using a Superdex 200 column (10/300 GL). (d) Native PAGE analysis of GFP oligomer charge variants with net charges of −3, −5, −7, −9 and −15. Enhanced solubility and gel separation of the oligomers are indicated.

Figure 2. Size-distribution analysis and visualization of discrete GFP polygons.

(a) Native PAGE analysis of purified GFP polygons from dimer to decamer. The gel was analysed by a fluorescent image analyser (top) and Coomassie blue staining (bottom). (b) SEC analysis of discrete GFP polygons from monomer to decamer. Absorbance spectra (at 488 nm) were normalized on their maximum peaks. (c) Representative TEM images and schematic drawings of discrete GFP polygons. Polygonal GFP arrangements in TEM images (the first row) were outlined by dotted yellow lines. Scale bar, 10 nm.

Figure 3. Multivalent display of functional proteins on GFP polygons.

(a) Schematic drawing of the triangular GFP oligomer with locations of N and C termini. (b) Native PAGE analysis of MBP-, protein G- and mCherry-displayed polygons. The gel was analysed by a fluorescent image analyser with 470-nm excitation and 530-nm emission filters (left), and 625-nm excitation and 695-nm emission filters (right). (c) Native PAGE analysis of discrete protein G polygons from dimer to decamer. (d) Schematic drawing and TEM images of N- (left set of panels) and C- (right set of panels) terminal-fused MBP polygons from dimer to tetramer. MBP and GFP are shown respectively in grey and green. Protein arrangements in TEM images (copies of those in the first column) were indicated with dotted yellow circles. Scale bars, 10 nm.

Figure 4. Fabrication and characterization of linearly opened GFP oligomers.

(a) Schematic representation of the construction of linearly opened GFP oligomers. CapGFP is designed to contain the GFP 11 strand connected to the N terminus of full-length GFP (1–11) with a His tag. Both CapGFP and GFP monomer (without His tag) were co-expressed in cells and opened oligomers were purified by His-affinity purifications. (b) Native PAGE analysis of open and circular forms of GFP oligomers. (c) Analysis of discrete opened oligomers from dimer to pentadecamer by native PAGE. (d) In-vitro assemblies of opened GFP oligomers with the GFP 1–10 fragment. Linearly opened GFP trimer, tetramer and pentamer were reacted with excess GFP 1–10 and resulting protein assemblies were analysed in a native PAGE gel. (e) TEM images of opened GFP trimer and tetramer. (f) TEM images and schematic drawing of MBP-displayed opened GFP trimer. A possible protein arrangement in a representative TEM image (a copy of the first image) was suggested with dotted yellow circles. Scale bars, 10 nm.

Figure 5. Multivalent interactions of protein G-functionalized GFP polygons.

(a) Schematic representation of the multivalent interactions between surface-bound antibodies and protein G polygons. Multivalent protein G polygons (dimer to heptamer) and monomeric protein G-GFP were applied on the surface-bound antibodies. (b,c) SPR responses on the associations (180 s) and dissociations (320 s) of multivalent protein G polygons against human (b) or mouse (c) antibodies. Constant mass concentrations of protein G polygons (5 μg ml⁻¹ for human and 10 μg ml⁻¹ for mouse antibodies) were used to maintain constant concentrations of the protein G unit, regardless of the valency. (d) Schematic representation of the interactions of surface-bound antibodies with protein G polygons (mono-, di- and trimers) or with genetically fused protein G (mono-,di- and trivalent) repeats. (e,f) SPR responses on the associations (180 s) and dissociations (320 s) of genetically fused protein G repeats (e) or protein G polygons (f) against mouse antibodies. All binding curves were normalized by subtracting the reflective index changes on sample injections.

Figure 6. Receptor clustering and internalization by protein G-functionalized GFP polygons.

(a) Confocal microscopy analysis of enhanced internalization of antibody–receptor clusters by protein G polygons. Cy5-labelled Erbitux (targeting EGFR) was treated to A549 cells with or without subsequent protein G hexamer treatment at 4 °C. After temperature change to 37 °C, receptor internalization was monitored by imaging Erbitux (Cy5) as well as protein G hexamer (GFP) at different time points. Cy5-Erbitux, red; Protein G-hexamer, green. (b) Flow cytometry analysis of antibody-mediated receptor internalization. A549 cells were treated with Cy5-Erbitux and subsequently protein G polygons with various valencies. Internalized levels of Cy5-Erbitux were quantified by flow cytometry. The error bars correspond to the s.e.m. of three independent measurements.