Protein assembly systems in natural and synthetic biology

Abstract

The traditional view of protein aggregation as being strictly disease-related has been challenged by many examples of cellular aggregates that regulate beneficial biological functions. When coupled with the emerging view that many regulatory proteins undergo phase separation to form dynamic cellular compartments, it has become clear that supramolecular assembly plays wide-ranging and critical roles in cellular regulation. This presents opportunities to develop new tools to probe and illuminate this biology, and to harness the unique properties of these self-assembling systems for synthetic biology for the purposeful manipulation of biological function.

Fig. 1

Overview of higher-order assemblies. a Protein assemblies display a spectrum of material properties, from solid-like amyloid fibers to highly dynamic liquid droplets. Examples of assemblies are shown below the spectrum. Highly stable assemblies include MAVS (mitochondrial antiviral signaling) protein fibers and Aβ (amyloid β) peptide amyloid fibrils. Highly dynamic assemblies include nucleoli, membraneless organelles with liquid-like shell around a more organized rigid core. The yeast prion protein Sup35 can convert between different structures: it constructs stable amyloid fibrils in its prion conformation and undergoes reversible gel formation under pH stress. Stress granules and P-bodies can also exist in different states, depending on the physiology of the cell. b Prions are self-propagating protein conformations. The prion conformation (purple) serves as a template to convert the soluble (gray) conformation into the prion conformation, which usually results in the growth of amyloid aggregates. The aggregates are fragmented by chaperone proteins, producing seeds that can nucleate the conformational conversion

Fig. 2

Protein assemblies play important roles in a variety of critical cellular processes. a In eukaryotic transcription, co-activators and transcription (txn.) factors form highly dynamic protein condensates that recruit RNA polymerase II (RNA pol II) and drive robust gene activation. b RNA-binding proteins (RBPs) and RNAs coalesce to form RNP granules, which serve different RNA processing functions, such as mRNA storage and degradation, ribosome biogenesis, and localized translation. In one intriguing example, prion-like aggregation of CPEB3 promotes translation in activated synapses to potentiate long-term memory. c Higher-order assemblies play key roles in innate immunity. For example, prion-like polymerization of the MAVS adaptor protein in response to viral infection leads to amplification and stabilization of the antiviral response. d In yeast, stochastic switching between [prion⁻] and [PRION⁺] states in a population of cells enables phenotypic diversification and may promote survival in uncertain environments. Figure adapted from Fig. 1B in [136]. In prion nomenclature, brackets denote non-Mendalian inheritance and capital letters denote dominance in crosses

Fig. 3

Application of higher-order protein assembly in synthetic biology. a Synthetic membraneless organelles, formed using proteins that undergo phase separation, can be used to enforce orthogonality of regulatory connections and biochemical reactions. This principle was recently used to create synthetic orthogonally translating (OT) organelles as sites for producing proteins that incorporate unnatural amino acids. b Exacting control over the formation of intracellular protein assemblies using optoDroplets. In this scheme, IDRs fused to light-inducible oligomerization domains enable the induction of phase separation by illumination with light. c Protein assembly systems as the basis of sensing and signal processing devices. Left: Protein assemblies can undergo dramatic changes in structure in response to small variations in environmental conditions, enabling exquisite sensing capabilities. Right: Changes in aggregation can be used to control downstream cellular processes. In the yTRAP system, the solubility state of an assembly domain is coupled to the activity of a synthetic TF and consequent activation of GOIs. d Prion proteins can exist stably in distinct conformational states, offering the potential to create synthetic memory devices based on prion switching