Self-assembly coupled to liquid-liquid phase separation.

Liquid condensate droplets with distinct compositions of proteins and nucleic acids are widespread in biological cells. While it is known that such droplets, or compartments, can regulate irreversible protein aggregation, their effect on reversible self-assembly remains largely unexplored. In this article, we use kinetic theory and solution thermodynamics to investigate the effect of liquid-liquid phase separation on the reversible self-assembly of structures with well-defined sizes and architectures. We find that, when assembling subunits preferentially partition into liquid compartments, robustness against kinetic traps and maximum achievable assembly rates can be significantly increased. In particular, both the range of solution conditions leading to productive assembly and the corresponding assembly rates can increase by orders of magnitude. We analyze the rate equation predictions using simple scaling estimates to identify effects of liquid-liquid phase separation as a function of relevant control parameters. These results may elucidate self-assembly processes that underlie normal cellular functions or pathogenesis, and suggest strategies for designing efficient bottom-up assembly for nanomaterials applications.

Multiscale Modeling of Hepatitis B Virus Capsid Assembly and Its Dimorphism.

Hepatitis B virus (HBV) is an endemic, chronic virus that leads to 800000 deaths per year. Central to the HBV lifecycle, the viral core has a protein capsid assembled from many copies of a single protein. The capsid protein adopts different (quasi-equivalent) conformations to form icosahedral capsids containing 180 or 240 proteins: T = 3 or T = 4, respectively, in Caspar-Klug nomenclature. HBV capsid assembly has become an important target for recently developed antivirals; nonetheless, the assembly pathways and mechanisms that control HBV dimorphism remain unclear. We describe computer simulations of the HBV assembly, using a coarse-grained model that has parameters learned from all-atom molecular dynamics simulations of a complete HBV capsid and yet is computationally tractable. Dynamical simulations with the resulting model reproduce experimental observations of HBV assembly pathways and products. By constructing Markov state models and employing transition path theory, we identify pathways leading to T = 3, T = 4, and other experimentally observed capsid morphologies. The analysis shows that capsid polymorphism is promoted by the low HBV capsid bending modulus, where the key factors controlling polymorphism are the conformational energy landscape and protein-protein binding affinities.

Microcompartment assembly around multicomponent fluid cargoes.

This article describes dynamical simulations of the assembly of an icosahedral protein shell around a bicomponent fluid cargo. Our simulations are motivated by bacterial microcompartments, which are protein shells found in bacteria that assemble around a complex of enzymes and other components involved in certain metabolic processes. The simulations demonstrate that the relative interaction strengths among the different cargo species play a key role in determining the amount of each species that is encapsulated, their spatial organization, and the nature of the shell assembly pathways. However, the shell protein-shell protein and shell protein-cargo component interactions that help drive assembly and encapsulation also influence cargo composition within certain parameter regimes. These behaviors are governed by a combination of thermodynamic and kinetic effects. In addition to elucidating how natural microcompartments encapsulate multiple components involved within reaction cascades, these results have implications for efforts in synthetic biology to colocalize alternative sets of molecules within microcompartments to accelerate specific reactions. More broadly, the results suggest that coupling between self-assembly and multicomponent liquid-liquid phase separation may play a role in the organization of the cellular cytoplasm.

Thermodynamic Size Control in Curvature-Frustrated Tubules: Self-Limitation with Open Boundaries.

We use computational modeling to investigate the assembly thermodynamics of a particle-based model for geometrically frustrated assembly, in which the local packing geometry of subunits is incompatible with uniform, strain-free large-scale assembly. The model considers discrete triangular subunits that drive assembly toward a closed, hexagonal-ordered tubule, but have geometries that locally favor negative Gaussian curvature. We use dynamical Monte Carlo simulations and enhanced sampling methods to compute the free energy landscape and corresponding self-assembly behavior as a function of experimentally accessible parameters that control assembly driving forces and the magnitude of frustration. The results determine the parameter range where finite-temperature self-limiting assembly occurs, in which the equilibrium assembly size distribution is sharply peaked around a well-defined finite size. The simulations also identify two mechanisms by which the system can escape frustration and assemble to unlimited size, and determine the particle-scale properties of subunits that suppress unbounded growth.

Mechanisms of Scaffold-Mediated Microcompartment Assembly and Size Control.

This article describes a theoretical and computational study of the dynamical assembly of a protein shell around a complex consisting of many cargo molecules and long, flexible scaffold molecules. Our study is motivated by bacterial microcompartments, which are proteinaceous organelles that assemble around a condensed droplet of enzymes and reactants. As in many examples of cytoplasmic liquid-liquid phase separation, condensation of the microcompartment interior cargo is driven by flexible scaffold proteins that have weak multivalent interactions with the cargo. Our results predict that the shell size, amount of encapsulated cargo, and assembly pathways depend sensitively on properties of the scaffold, including its length and valency of scaffold-cargo interactions. Moreover, the ability of self-assembling protein shells to change their size to accommodate scaffold molecules of different lengths depends crucially on whether the spontaneous curvature radius of the protein shell is smaller or larger than a characteristic elastic length scale of the shell. Beyond natural microcompartments, these results have important implications for synthetic biology efforts to target alternative molecules for encapsulation by microcompartments or viral shells. More broadly, the results elucidate how cells exploit coupling between self-assembly and liquid-liquid phase separation to organize their interiors.

The role of the encapsulated cargo in microcompartment assembly.

Bacterial microcompartments are large, roughly icosahedral shells that assemble around enzymes and reactants involved in certain metabolic pathways in bacteria. Motivated by microcompartment assembly, we use coarse-grained computational and theoretical modeling to study the factors that control the size and morphology of a protein shell assembling around hundreds to thousands of molecules. We perform dynamical simulations of shell assembly in the presence and absence of cargo over a range of interaction strengths, subunit and cargo stoichiometries, and the shell spontaneous curvature. Depending on these parameters, we find that the presence of a cargo can either increase or decrease the size of a shell relative to its intrinsic spontaneous curvature, as seen in recent experiments. These features are controlled by a balance of kinetic and thermodynamic effects, and the shell size is assembly pathway dependent. We discuss implications of these results for synthetic biology efforts to target new enzymes to microcompartment interiors.

Many-molecule encapsulation by an icosahedral shell.

We computationally study how an icosahedral shell assembles around hundreds of molecules. Such a process occurs during the formation of the carboxysome, a bacterial microcompartment that assembles around many copies of the enzymes ribulose 1,5-bisphosphate carboxylase/ oxygenase and carbonic anhydrase to facilitate carbon fixation in cyanobacteria. Our simulations identify two classes of assembly pathways leading to encapsulation of many-molecule cargoes. In one, shell assembly proceeds concomitantly with cargo condensation. In the other, the cargo first forms a dense globule; then, shell proteins assemble around and bud from the condensed cargo complex. Although the model is simplified, the simulations predict intermediates and closure mechanisms not accessible in experiments, and show how assembly can be tuned between these two pathways by modulating protein interactions. In addition to elucidating assembly pathways and critical control parameters for microcompartment assembly, our results may guide the reengineering of viruses as nanoreactors that self-assemble around their reactants.