Principles Governing the Self-Assembly of Coiled-Coil Protein Nanoparticles

Self-assembly refers to the spontaneous organization of individual building blocks into higher order structures. It occurs in biological systems such as spherical viruses, which utilize icosahedral symmetry as a guiding principle for the assembly of coat proteins into a capsid shell. In this study, we characterize the self-assembling protein nanoparticle (SAPN) system, which was inspired by such viruses. To facilitate self-assembly, monomeric building blocks have been designed to contain two oligomerization domains. An N-terminal pentameric coiled-coil domain is linked to a C-terminal coiled-coil trimer by two glycine residues. By combining monomers with inherent propensity to form five- and threefold symmetries in higher order agglomerates, the supposition is that nanoparticles will form that exhibit local and global symmetry axes of order 3 and 5. This article explores the principles that govern the assembly of such a system. Specifically, we show that the system predominantly forms according to a spherical core-shell morphology using a combination of scanning transmission electron microscopy and small angle neutron scattering. We introduce a mathematical toolkit to provide a specific description of the possible SAPN morphologies, and we apply it to characterize all particles with maximal symmetry. In particular, we present schematics that define the relative positions of all individual chains in the symmetric SAPN particles, and provide a guide of how this approach can be generalized to nonspherical morphologies, hence providing unprecedented insights into their geometries that can be exploited in future applications

Effects of Protein Conformation, Apparent Solubility, and Protein–Protein Interactions on the Rates and Mechanisms of Aggregation for an IgG1Monoclonal Antibody

Non-native protein aggregation is a key degradation pathway of immunoglobulins. In this work, the aggregation kinetics of an immunoglobulin gamma-1 monoclonal antibody (IgG1 mAb) in different solution environments was monitored over a range of incubation temperatures for up to seven months using size exclusion chromatography. Histidine and citrate buffers with/without sodium chloride were employed to modulate the mAb’s conformational stability, solubility (in the presence of polyethylene glycol, PEG), and protein–protein interactions as measured by differential scanning calorimetry, PEG precipitation, and static light scattering, respectively. The effect of these parameters on the mechanism(s) of mAb aggregation during storage at different temperatures was determined using kinetic models, which were used to fit aggregation data to determine rate constants for aggregate nucleation and growth processes. This approach was used to investigate the effects of colloidal protein–protein interactions and solubility values (in PEG solutions) on the mechanisms and rates of IgG1 mAb aggregation as a function of temperature-induced structural perturbations. Aggregate nucleation and growth pathways for this IgG1 mAb were sensitive to temperature and overall conformational stability. Aggregate growth, on the other hand, was also sensitive to conditions affecting the solubility of the mAb, particularly at elevated temperatures

Dot Blot analysis of different chimeric NPs displaying βbarrel for the detection of exposed antigen.

(1) βbarrel-ferritin, (2) βbarrel-mI3, (3) βbarrel-Encapsulin, (4) βbarrel-CP3, (5) βbarrel-HBcAg, (NC) Negative control represented by naked ferritin, (PC) Monomeric βbarrel used as positive control.</p

TEM analysis of unstructured NPs.

(A) βbarrel-Qbeta (B) βbarrel-encapsulin. Only aggregates or monomers were detected after affinity and size exclusion chromatography. (TIF)</p

Principles Governing the Self-Assembly of Coiled-Coil Protein Nanoparticles

Self-assembly refers to the spontaneous organization of individual building blocks into higher order structures. It occurs in biological systems such as spherical viruses, which utilize icosahedral symmetry as a guiding principle for the assembly of coat proteins into a capsid shell. In this study, we characterize the self-assembling protein nanoparticle (SAPN) system, which was inspired by such viruses. To facilitate self-assembly, monomeric building blocks have been designed to contain two oligomerization domains. An N-terminal pentameric coiled-coil domain is linked to a C-terminal coiled-coil trimer by two glycine residues. By combining monomers with inherent propensity to form five- and threefold symmetries in higher order agglomerates, the supposition is that nanoparticles will form that exhibit local and global symmetry axes of order 3 and 5. This article explores the principles that govern the assembly of such a system. Specifically, we show that the system predominantly forms according to a spherical core-shell morphology using a combination of scanning transmission electron microscopy and small angle neutron scattering. We introduce a mathematical toolkit to provide a specific description of the possible SAPN morphologies, and we apply it to characterize all particles with maximal symmetry. In particular, we present schematics that define the relative positions of all individual chains in the symmetric SAPN particles, and provide a guide of how this approach can be generalized to nonspherical morphologies, hence providing unprecedented insights into their geometries that can be exploited in future applications

Predicted 3D model of Encapsulin (A) and HBcAg (B) NPs displaying βbarrel into an exposed loop.

(PNG)</p

Negative staining transmission electron microscopy (NSTEM) of chimeric NPs displaying βbarrel antigen after SEC purification.

Properly assembled particles were detected for (A) βbarrel-Ferritin with a diameter of 25nm (B) βbarrel-mI3 with a diameter of 30nm (C) βbarrel-Encapsulin presents a diameter of 30nm (D) βbarrel-CP3 presents a diameter of 30nm (E) βbarrel-HBcAg with a diameter of 35nm. Scale bars inserted in the pictures correspond to 50nm (A-B-E) and 100nm (C-D).</p

Summary of protein-based nanoparticles and virus like particles tested in this study.

Summary of protein-based nanoparticles and virus like particles tested in this study.</p

Structural analysis and in silico design of chimeric NPs.

(A) Cartoon representation of 3D structure of fHbp antigen (pdb code 3KVD). In grey it is reported the N-terminal domain (residues 1–118) while in yellow it is shown the C-terminal βbarrel domain used in this work (residues 119–249). (B) Cartoon representation of the monomeric structure of each tested NPs. Engineerable sites explored for the genetic fusion of the antigen are highlighted: the N terminus in dark blue and the exposed loops in red. (C) Cartoon of predicted 3D models of each chimera obtained with Rosetta homology modelling. The βbarrell exposed was represented in yellow. Images were obtained with ChimeraX (panel A, C) and Pymol (panel B).</p

S1 Raw images -

(PDF)</p