Pulsed electric fields induce modulation of protein liquid-liquid phase separation.

The time-resolved dynamic assembly and the structures of protein liquid dense clusters (LDCs) were analyzed under pulsed electric fields (EFs) applying complementary polarized and depolarized dynamic light scattering (DLS/DDLS), optical microscopy, and transmission electron microscopy (TEM). We discovered that pulsed EFs substantially affected overall morphologies and spatial distributions of protein LDCs and microcrystals, and affected the phase diagrams of LDC formation, including enabling protein solutions to overcome the diffusive flux energy barrier to phase separate. Data obtained from DLS/DDLS and TEM showed that LDCs appeared as precursors of protein crystal nuclei, followed by the formation of ordered structures within LDCs applying a pulsed EF. Experimental results of circular dichroism spectroscopy provided evidence that the protein secondary structure content is changing under EFs, which may consequently modulate protein-protein interactions, and the morphology, dimensions, and internal structure of LDCs. Data and results obtained unveil options to modulate the phase diagram of crystallization, and physical morphologies of protein LDCs and microcrystals by irradiating sample suspensions with pulsed EFs.