Programmable Assembly of Elastin-like Polypeptides Within Droplet Microenvironments

Disordered proteins lack a defined secondary and higher order structure, possess a large number of biological functions, and are prevalent throughout nature. One important property of certain disordered proteins in biology is an ability to phase separate within the interior of a cell and form liquid non-membrane bound organelles. These compartments are thought to play crucial roles in various cellular processes such as ribonucleic acid maintenance and gene expression. While the physicochemical drivers and biophysical properties of intracellular organelles is becoming clear, their functions remain poorly understood due to the difficulty in probing them and the lack of engineered models of them.

This work addresses the need for engineered systems of membraneless organelles and consists of programming the assembly of elastin-like polypeptides, an archetypal class of disordered proteins, into model organelles within droplet microenvironments. We synthesized a library of recombinant elastin-like polypeptides, and studied their phase separation and assembly into various organelles, from layered to mixed to size-controlled, within water microdroplets using lightfield, darkfield, confocal, and fluorescence microscopy. We coupled this with bulk characterization techniques, in the form of spectrophotometry and light scattering, to develop a theoretical framework for understanding and predicting disordered protein phase separation into these biologically relevant structures. Furthermore, we adapted our findings to engineer new thermoresponsive colloidal gels with size tunable across four orders of magnitude (nano-to-meso-to-microscale) that are comprised of elastin-like polypeptides containing unnatural amino acid photocrosslinkers. Lastly, we developed model functional ribonucleoprotein organelles composed of RNA-binding elastin-like polypeptides designed de novo, and show that these organelles temporally regulate gene expression within droplet-based protocells. Taken together, this body of work offers new insights into (1) our understanding of the genetic to molecular to macroscale relationships encoding naturally occurring intrinsically disordered protein assemblies, (2) the design rules of these assemblies in cell biology, and it enables (3) the facile engineering of unprecedented polypeptide biomaterials in the form of (i) thermoresponsive photocrosslinked colloidal gels with potential utility in drug delivery and (ii) functional, artificial ribonucleoprotein granules that lay the foundation for developing more complex, realistic artificial cells.