Protein cage assembly across multiple length scales.

Within the materials science community, proteins with cage-like architectures are being developed as versatile nanoscale platforms for use in protein nanotechnology. Much effort has been focused on the functionalization of protein cages with biological and non-biological moieties to bring about new properties of not only individual protein cages, but collective bulk-scale assemblies of protein cages. In this review, we report on the current understanding of protein cage assembly, both of the cages themselves from individual subunits, and the assembly of the individual protein cages into higher order structures. We start by discussing the key properties of natural protein cages (for example: size, shape and structure) followed by a review of some of the mechanisms of protein cage assembly and the factors that influence it. We then explore the current approaches for functionalizing protein cages, on the interior or exterior surfaces of the capsids. Lastly, we explore the emerging area of higher order assemblies created from individual protein cages and their potential for new and exciting collective properties.

Experimental models for dynamic compartmentalization of biomolecules in liquid organelles: Reversible formation and partitioning in aqueous biphasic systems.

Living cells contain numerous subcellular compartments, many of which lack membranous boundaries and are thought to occur due to liquid-liquid phase coexistence. This review will introduce these biological membraneless organelles and discuss simple experimental models based on liquid-liquid phase separation in polymer solutions. When more than one phase is present, solutes such as proteins or nucleic acids can be compartmentalized by partitioning into one of the phases. This could confer benefits to the cell such as enhanced reaction rates or sequestration of toxic molecules. Liquid-like compartments inside living cells are often dynamic, for example, appearing and disappearing in response to stimuli and/or at different points in the cell cycle. We will discuss mechanisms by which phase transitions can be induced in the laboratory and inside living cells, with special emphasis on regulating phase formation by phosphorylation state. This work is motivated by a desire to understand the physical and chemical mechanisms that underlie biological processes and to enable new nonbiological applications.

RNA-Based Coacervates as a Model for Membraneless Organelles: Formation, Properties, and Interfacial Liposome Assembly.

Liquid-liquid phase separation is responsible for formation of P granules, nucleoli, and other membraneless subcellular organelles composed of RNA and proteins. Efforts to understand the physical basis of liquid organelle formation have thus far focused on intrinsically disordered proteins (IDPs) as major components that dictate occurrence and properties. Here, we show that complex coacervates composed of low complexity RNA (polyuridylic acid, polyU) and short polyamines (spermine and spermidine) share many features of IDP-based coacervates. PolyU/polyamine coacervates compartmentalize biomolecules (peptides, oligonucleotides) in a sequence- and length-dependent manner. These solutes retain mobility within the coacervate droplets, as demonstrated by rapid recovery from photobleaching. Coacervation is reversible with changes in solution temperature due to changes in the polyU structure that impact its interactions with polyamines. We further demonstrate that lipid vesicles assemble at the droplet interface without impeding RNA entry/egress. These vesicles remain intact at the interface and can be released upon temperature-induced droplet dissolution.

Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles.

Biological cells are highly organized, with numerous subcellular compartments. Phosphorylation has been hypothesized as a means to control the assembly/disassembly of liquid-like RNA- and protein-rich intracellular bodies, or liquid organelles, that lack delimiting membranes. Here, we demonstrate that charge-mediated phase separation, or complex coacervation, of RNAs with cationic peptides can generate simple model liquid organelles capable of reversibly compartmentalizing biomolecules. Formation and dissolution of these liquid bodies was controlled by changes in peptide phosphorylation state using a kinase/phosphatase enzyme pair. The droplet-generating phase transition responded to modification of even a single serine residue. Electrostatic interactions between the short cationic peptides and the much longer polyanionic RNAs drove phase separation. Coacervates were also formed on silica beads, a primitive model for localization at specific intracellular sites. This work supports phosphoregulation of complex coacervation as a viable mechanism for dynamic intracellular compartmentalization in membraneless organelles.

Colocalization and Sequential Enzyme Activity in Aqueous Biphasic Systems: Experiments and Modeling.

Subcellular compartmentalization of biomolecules and their reactions is common in biology and provides a general strategy for improving and/or controlling kinetics in metabolic pathways that contain multiple sequential enzymes. Enzymes can be colocalized in multiprotein complexes, on scaffolds or inside subcellular organelles. Liquid organelles formed by intracellular phase coexistence could provide an additional means of sequential enzyme colocalization. Here we use experiment and computation to explore the kinetic consequences of sequential enzyme compartmentalization into model liquid organelles in a crowded polymer solution. Two proteins of the de novo purine biosynthesis pathway, ASL (adenylosuccinate lyase, Step 8) and ATIC (5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase, Steps 9 and 10), were studied in a polyethylene glycol/dextran aqueous two-phase system. Dextran-rich phase droplets served as model liquid compartments for enzyme colocalization. In this system, which lacks any specific binding interactions between the phase-forming polymers and the enzymes, we did not observe significant rate enhancements from colocalization for the overall reaction under our experimental conditions. The experimental results were used to adapt a mathematical model to quantitatively describe the kinetics. The mathematical model was then used to explore additional, experimentally inaccessible conditions to predict when increased local concentrations of enzymes and substrates can (or cannot) be expected to yield increased rates of product formation. Our findings indicate that colocalization within these simplified model liquid organelles can lead to enhanced metabolic rates under some conditions, but that very strong partitioning into the phase that serves as the compartment is necessary. In vivo, this could be provided by specific binding affinities between components of the liquid compartment and the molecules to be localized within it.

Interactions of macromolecular crowding agents and cosolutes with small-molecule substrates: effect on horseradish peroxidase activity with two different substrates.

The importance of solution composition on enzymatic reactions is increasingly appreciated, particularly with respect to macromolecular cosolutes. Macromolecular crowding and its effect on enzymatic reactions has been studied for several enzymes and is often understood in terms of changes to enzyme conformation. Comparatively little attention has been paid to the chemical properties of small-molecule substrates for enzyme reactions in crowded solution. In this article, we studied the reaction of horseradish peroxidase (HRP) with two small-molecule substrates that differ in their hydrophobicity. Crowding agents and cosolutes had quite different effects on HRP activity when the substrate used was 3,3',5,5'-tetramethylbenzidine (TMB, which is hydrophobic) as compared to o-phenylenediamine (OPD, which is more hydrophilic). Reaction rates with TMB were much more sensitive to the presence of crowding agents and cosolutes than OPD, suggesting that the small-molecule substrates may themselves be interacting with crowders and cosolutes. At high polyethylene glycol (PEG) concentrations (25-30 wt/wt %), no reaction was observed for TMB. Even at lower concentrations, Michaelis constants (KM) for HRP with the more hydrophobic substrate increased in the presence of crowding agents and cosolutes, particularly with PEG. Diffusion of TMB and OPD in the PEG and dextran reaction media was evaluated using pulsed field gradient nuclear magnetic resonance (PFG-NMR). The diffusivity of the TMB decreased 3.9× in 10% PEG 8k compared to that in buffer and decreased only 1.7× for OPD. Together, these data suggest that weak attractive interactions between small-molecule substrates and crowders or cosolutes can reduce substrate chemical activity and consequently decrease enzyme activity and that these effects vary with the identity of the molecules involved. Because many enzymes can act on multiple substrates, it is important to consider substrate chemistry in understanding enzymatic reactions in complex media such as biological fluids.

Coupled enzyme reactions performed in heterogeneous reaction media: experiments and modeling for glucose oxidase and horseradish peroxidase in a PEG/citrate aqueous two-phase system.

The intracellular environment in which biological reactions occur is crowded with macromolecules and subdivided into microenvironments that differ in both physical properties and chemical composition. The work described here combines experimental and computational model systems to help understand the consequences of this heterogeneous reaction media on the outcome of coupled enzyme reactions. Our experimental model system for solution heterogeneity is a biphasic polyethylene glycol (PEG)/sodium citrate aqueous mixture that provides coexisting PEG-rich and citrate-rich phases. Reaction kinetics for the coupled enzyme reaction between glucose oxidase (GOX) and horseradish peroxidase (HRP) were measured in the PEG/citrate aqueous two-phase system (ATPS). Enzyme kinetics differed between the two phases, particularly for the HRP. Both enzymes, as well as the substrates glucose and H2O2, partitioned to the citrate-rich phase; however, the Amplex Red substrate necessary to complete the sequential reaction partitioned strongly to the PEG-rich phase. Reactions in ATPS were quantitatively described by a mathematical model that incorporated measured partitioning and kinetic parameters. The model was then extended to new reaction conditions, i.e., higher enzyme concentration. Both experimental and computational results suggest mass transfer across the interface is vital to maintain the observed rate of product formation, which may be a means of metabolic regulation in vivo. Although outcomes for a specific system will depend on the particulars of the enzyme reactions and the microenvironments, this work demonstrates how coupled enzymatic reactions in complex, heterogeneous media can be understood in terms of a mathematical model.

Phase separation as a possible means of nuclear compartmentalization.

The nucleus is perhaps the most familiar organelle within eukaryotic cells, serving as a compartment to house the genetic material. The nuclear volume is subdivided into a variety of functional and dynamic nuclear bodies not separated from the nucleoplasm by membranes. It has been hypothesized that aqueous phase separation brought about by macromolecular crowding may be in part responsible for these intranuclear compartments. This chapter discusses macromolecular solution chemistry with regard to several common types of phase separation in polymer solutions as well as to recent evidence that suggests that cytoplasmic and nuclear bodies may exist as liquid phases. We then examine the functional significance of phase separation and how it may serve as a means of compartmentalizing various nuclear activities, and describe recent studies that have used simple model systems to generate coexisting aqueous phase compartments, concentrate molecules within them, and perform localized biochemical reactions.