Engineering reversibility of liquid-liquid phase separation in artificial proteins

Small-angle X-ray photon correlation spectroscopy (SA-XPCS) reveals the impact of elastin-like polypeptide (ELP) length on the reversibility of temperature-induced liquid-liquid phase separation (LLPS). The sensitivity of SA-XPCS to subtle energy landscape changes allows probing of the complex LLPS behavior in ELPs. We observe irreversible LLPS in (VPAVG)₁₀​, while (VPAVG)₃₀​ shows partial reversibility upon cooling and complete reversal after extended incubation at 6^oC. A universal two-step decay in dynamics is observed across all ELPs, suggesting a transition from fast diffusion of microscopic droplets to arrested coarsening of a gel network. This transition is consistent across ELPs of different lengths, despite variations in phase separation temperatures, structure formation rates, and reversibility characteristics. In addition to the LLPS studies, a fully robotic SA-XPCS protocol is developed to eliminate manual handling-induced heating for future studies. The 100x increase in coherent flux from the APS upgrade will enable studies at near-physiological concentrations, elucidating the role of LLPS in cellular function and informing the design of bio-inspired materials. Furthermore, the significant increase of the measurement throughput as a result of the coherent flux boost will pave the way to autonomous design of functioning bio-materials through the development of beamline automation and AI tool suites.