Liquid-liquid phase separation (LLPS) has recently been proposed as a feature of the silk spinning process in spiders and silkworms.<sup>1</sup> LLPS of proteins is known to occur in response to external stimuli such as changes in pH, temperature, and various salt ions and is important for stabilizing intrinsically disordered proteins (IDPs) in dense liquid droplets at high concentrations. Sometimes, however, these droplets become destabilized, causing IDPs to form fibers irreversibly. While this method of fiber formation is interesting from the perspective of biomaterials research, it has also been observed in proteins involved in neurodegenerative disease, including tau (Alzheimer’s disease), huntingtin (Huntington’s disease), and islet amyloid polypeptide (type II diabetes), yet the details surrounding stabilization of IDPs by LLPS and the factors that lead to fibrillization are still not well understood. Using spider silk as a model system, this study focuses on development of proteins in LLPS in the context of biomaterials formation using solution Nuclear Magnetic Resonance (NMR) spectroscopy and electron microscopy (EM). While solution NMR provides information regarding dynamics and intra- and intermolecular atomic level interactions between IDPs, EM gives insight into the mesoscale development of proteins as they transition from dense droplets to solid fibers. This work should be of broad interest to those investigating IDPs, biomaterials and the fundamental physical chemistry, and structural biology of LLPS.

Probing the liquid-liquid phase separation of spider silk proteins with solution NMR and electron microscopy

ACS Spring 2022

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