Fluctuating nonlinear spring theory: Strength, deformability, and toughness of biological nanoparticles from theoretical reconstruction of force-deformation spectra.

We developed the Fluctuating Nonlinear Spring (FNS) model to describe the dynamics of mechanical deformation of biological particles, such as virus capsids. The theory interprets the force-deformation spectra in terms of the "Hertzian stiffness" (non-linear regime of a particle's small-amplitude deformations), elastic constant (large-amplitude elastic deformations), and force range in which the particle's fracture occurs. The FNS theory enables one to quantify the particles' elasticity (Young's moduli for Hertzian and bending deformations), and the limits of their strength (critical forces, fracture toughness) and deformability (critical deformations) as well as the probability distributions of these properties, and to calculate the free energy changes for the particle's Hertzian, elastic, and plastic deformations, and eventual fracture. We applied the FNS theory to describe the protein capsids of bacteriophage P22, Human Adenovirus, and Herpes Simplex virus characterized by deformations before fracture that did not exceed 10-19% of their size. These nanoshells are soft (~1-10-GPa elastic modulus), with low ~50-480-kPa toughness - a regime of material behavior that is not well understood, and with the strength increasing while toughness decreases with their size. The particles' fracture is stochastic, with the average values of critical forces, critical deformations, and fracture toughness comparable with their standard deviations. The FNS theory predicts 0.7-MJ/mol free energy for P22 capsid maturation, and it could be extended to describe uniaxial deformation of cylindrical microtubules and ellipsoidal cellular organelles.

Infectious bursal disease virus capsid assembly and maturation by structural rearrangements of a transient molecular switch.

Infectious bursal disease virus (IBDV), a double-stranded RNA (dsRNA) virus belonging to the Birnaviridae family, is an economically important avian pathogen. The IBDV capsid is based on a single-shelled T=13 lattice, and the only structural subunits are VP2 trimers. During capsid assembly, VP2 is synthesized as a protein precursor, called pVP2, whose 71-residue C-terminal end is proteolytically processed. The conformational flexibility of pVP2 is due to an amphipathic alpha-helix located at its C-terminal end. VP3, the other IBDV major structural protein that accomplishes numerous roles during the viral cycle, acts as a scaffolding protein required for assembly control. Here we address the molecular mechanism that defines the multimeric state of the capsid protein as hexamers or pentamers. We used a combination of three-dimensional cryo-electron microscopy maps at or close to subnanometer resolution with atomic models. Our studies suggest that the key polypeptide element, the C-terminal amphipathic alpha-helix, which acts as a transient conformational switch, is bound to the flexible VP2 C-terminal end. In addition, capsid protein oligomerization is also controlled by the progressive trimming of its C-terminal domain. The coordination of these molecular events correlates viral capsid assembly with different conformations of the amphipathic alpha-helix in the precursor capsid, as a five-alpha-helix bundle at the pentamers or an open star-like conformation at the hexamers. These results, reminiscent of the assembly pathway of positive single-stranded RNA viruses, such as nodavirus and tetravirus, add new insights into the evolutionary relationships of dsRNA viruses.

Double hexamer disruption and biochemical activities of Methanobacterium thermoautotrophicum MCM.

Methanobacterium thermoautotrophicum MCM (mtMCM) is a helicase required for DNA replication. Previous electron microscopy studies have shown mtMCM in several oligomeric forms. However, biochemical studies suggest that mtMCM is a dodecamer, likely a double hexamer (dHex). The crystal structure of the N-terminal fragment of mtMCM reveals a stable dHex architecture. To further confirm that the dHex is not an artifact of crystal packing of two hexamers, we investigated the relevance of the dHex by disrupting the hexamer-hexamer interactions seen in the crystal structure via site-directed mutagenesis and examining various biochemical activities of the mutants in vitro. Using a combination of biochemical and structural assays, we demonstrated that changing arginine to alanine at amino acid position 161 or the insertion of a six-aminoacid peptide at the hexamer-hexamer interface disrupted dHex formation and produced stable single hexamers (sHex). Furthermore, we showed that the sHex mutants retained wild-type level of ATPase and DNA binding activities but had decreased helicase activity when compared with the wild type dHex protein. These biochemical properties of mtMCM are reminiscent of those of SV40 large T antigen, suggesting that the dHex form of mtMCM may be the active helicase for DNA unwinding during the bidirectional DNA replication.