Liquid–liquid phase separation of tropoelastin has long been considered to be an important early step in the complex process of elastin fiber assembly in the body and has inspired the development of elastin-like peptides with a wide range of industrial and biomedical applications. Despite decades of study, the material state of the condensed liquid phase of elastin and its subsequent maturation remain poorly understood. Here, using a model minielastin that mimics the alternating domain structure of full-length tropoelastin, we examine the elastin liquid phase. We combine differential interference contrast (DIC), fluorescence, and scanning electron microscopy with particle-tracking microrheology to resolve the material transition occurring within elastin liquids over time in the absence of exogenous cross-linking. We find that this transition is accompanied by an intermediate stage marked by the coexistence of insoluble solid and dynamic liquid phases giving rise to significant spatial heterogeneities in material properties. We further demonstrate that varying the length of the terminal hydrophobic domains of minielastins can tune the maturation process. This work not only resolves an important step in the hierarchical assembly process of elastogenesis but further contributes mechanistic insight into the diverse repertoire of protein condensate maturation pathways with emerging importance across biology.

biochemistry is computationally predictable. Partial Order Optimum Likelihood (POOL) is a machine learning method developed by us to predict residues important for function, using the 3D structure of the query protein. The input features to POOL are based on computed electrostatic and chemical properties from THEMATICS. These input features are effectively measures of the strength of coupling between protonation events. POOL is used to predict the residues important for catalysis and ligand binding. Typical predicted catalytic sites are characterized by networks of strongly coupled protonation states; these networks impart the necessary electrostatic and proton-transfer properties to the active residues in the first layer around the reacting substrate molecule(s). Most often these networks include first-, second-, and sometimes third-layer residues. POOL-predicted, multi-layer active sites with significant participation by distal residues have been verified experimentally by single-point sitedirected mutagenesis and kinetics assays for multiple examples, including human phosphoglucose isomerase, human PARK2 (an E3 ubiquitin ligase), and E. coli ornithine transcarbamoylase. Mechanisms for the effects of diseaseassociated mutations, and implications for personalized medicine, are discussed.

Tropoelastin is a key protein in the formation of connective tissue such as lungs, arteries, and cartilage. The assembly and further cross‐linking process of tropoelastin culminates in the formation of elastin fibers, a resilient biomaterial capable of withstanding numerous cycles of stress and strain. Like other intrinsically disordered proteins, tropoelastin can undergo liquid‐liquid phase separation in vitro and in the extracellular space. This event is thought to aid in the self‐assembly and subsequent maturation of elastin fibers. Although the mechanical properties and morphology of mature elastin fibers have been extensively studied, the properties of elastin liquid droplets and their subsequent maturation into a solid remains poorly understood. Here, we use a model mini‐elastin polypeptide that mimics the domain architecture of naturally occurring tropoelastin to characterize this transition. We use fluorescence recovery after photobleaching (FRAP) and microrheology to capture the transition of elastin droplets from a liquid to a solid‐like state. We find that elastin droplets behave as viscous fluids at early incubation times, however, a rapid liquid‐to‐solid transition is observed in a timeframe of 80 minutes when held at constant temperature, even in the absence of cross‐linker. We further resolve the changes in dynamics, diffusion, and material properties of elastin condensates over the course of this transition. This work, which reveals the material transition from within elastin condensates, lends new insight into the early steps of the self‐assembly process of elastin while also contributing to the expanding repertoire of condensate maturation models in biological systems.