Researchers have captured the molecular-level self-assembly of a virus-like particle using single-molecule imaging. Published in Nature, this reveals a nucleated growth mechanism of viral capsids by tracking mi3-VLP (SC003-mi3) monomers through mass photometry, unlocking precise structural insights for biomedical engineering and nanotechnology applications.
Engineering the SC003-mi3 Subunit Architecture
Understanding how viral capsids build themselves requires absolute precision at the nanometer scale. The experimental framework relies on the mi3-VLP, also designated as SC003-mi3, derived from the i3-01 architecture. Researchers introduced two mutations into this sequence to eliminate surface-exposed cysteines. This modification prevents aggregation during the protein folding and purification phases.
Furthermore, a SpyCatcher domain was genetically fused to the N-terminus. This addition allows plug-and-play attachment of SpyTagged proteins. The modified plasmid, designated as pET28a-SpyCatcher003(SC003)-mi3, was originally supplied by Professor Mark Howarth from Cambridge University. Transformation into Escherichia coli BL21(DE3) RIPL cells kicked off the production pipeline. Cultures grown in Luria-Bertani media supplemented with 50 µg ml−1 kanamycin were induced with 0.42 mM IPTG at an optical density (OD600) of approximately 0.6.
Following a 16-hour expression run at 22 °C, high-pressure homogenization at 30,000 psi lysed the cellular biomass. Clarified supernatants underwent ammonium sulfate precipitation at 170 mg/ml to isolate the target macromolecular structures before subsequent chromatographic purification.
Disassembly and Mass Photometry Kinetics
To watch these protein cages construct themselves from scratch, researchers had to take them apart first. Mi3-VLPs can reversibly disassemble and reassemble when exposed to a 2.5 M concentration of guanidinium thiocyanate. The team prepared solutions containing purified mi3-VLPs at a monomer concentration of 84 µM, breaking them down across varying monomer ranges from 35 to 5.8 µM.
Initiating the assembly phase required a rapid 100-fold dilution into an assembly buffer containing 20 mM HEPES at pH 7.4 and 138 mM NaCl. This drop in denaturant concentration brought the final mi3-monomer concentration down to a window between 350 and 58 nM. After an equilibration period of 30 minutes at room temperature, the team quantified the mass distribution of the reassembled viral-like particles using a commercial mass photometer, specifically the TwoMP system manufactured by Refeyn Ltd.
Mass photometry changes the game for structural biology. Instead of relying on ensemble-averaging techniques that blur out transient intermediates, single-molecule mass photometry measures light scattering from individual molecules landing on a glass coverslip. This approach provides a direct readout of molecular weight distributions in real time.
Visualizing the Nucleated Growth Mechanism
The resulting data offers a look at capsid biogenesis. Single-molecule imaging confirms a nucleated growth mechanism, where initial formation requires a rate-limiting nucleation step before rapid elongation takes over. Once the nucleus forms, subunits add onto the growing capsid structure with remarkable efficiency.
Engineering robust protein cages demands exact oversight of ionic strength, pH, and protein concentration.
