Controlling the Biophysical Properties and Functionality of Phase-Separated Elastin-Based Droplets through Amino Acid Sequence Mutations

Abstract

Liquid-liquid phase separation underlies the spontaneous formation of protein-rich droplets in a protein-poor solution phase, concomitant with a sharp increase in solution turbidity. Phase separation is typically triggered in response to a stimulus such as change in protein concentration, ionic strength, or temperature, and may be reversible or precede gelation, fibre formation, or aggregation. Phase separation is a well-known property of synthetic polymers, and is exploited for material design, for example, capsules for drug delivery and fast-setting hydrogels, but is not well understood for proteins. Some examples of phase-separating protein-based systems include lens gamma crystallin (implicated in cataract formation), ‘membraneless organelles’ (transiently formed compartments that regulate molecular interactions), and self-assembling elastomeric proteins, including some spider silks, insect resilin and vertebrate elastin, for which phase separation is on-pathway for the formation of elastic materials. Here we studied the phase separation of model polypeptides based on the protein elastin. These polypeptides contain both regions of well-defined secondary structure and intrinsic disorder. Droplet growth and biophysical properties were monitored using a variety of microscopy and optical spectroscopy techniques. We report the effect of amino acid sequence mutations on the size, stability, reversibility and interactions of phase-separated droplets, and address implications for functional tunability, including loading of small molecules.