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Knowledge about the structural and dynamic properties of proteins that form membrane-less organelles in cells via liquid–liquid phase separation (LLPS) is required for understanding the process at a molecular level. We used spin labeling and electron paramagnetic resonance (EPR) spectroscopy to investigate the dynamic properties (rotational diffusion) of the low complexity N-terminal domain of cytoplasmic polyadenylation element binding-4 protein (CPEB4NTD) across its LLPS transition, which takes place with increasing temperature. We report the coexistence of three spin labeled CPEB4NTD (CPEB4*) populations with distinct dynamic properties representing different conformational spaces, both before and within the LLPS state. Monomeric CPEB4* exhibiting fast motion defines population I and shows low abundance prior to and following LLPS. Populations II and III are part of CPEB4* assemblies where II corresponds to loose conformations with intermediate range motions and population III represents compact conformations with strongly attenuated motions. As the temperature increased the population of component II increased reversibly at the expense of component III, indicating the existence of an III ⇌ II equilibrium. We correlated the macroscopic LLPS properties with the III ⇌ II exchange process upon varying temperature and CPEB4* and salt concentrations. We hypothesized that weak transient intermolecular interactions facilitated by component II lead to LLPS, with the small assemblies integrated within the droplets. The LLPS transition, however, was not associated with a clear discontinuity in the correlation times and populations of the three components. Importantly, CPEB4NTD exhibits LLPS properties where droplet formation occurs from a preformed microscopic assembly rather than the monomeric protein molecules.

Solid-state drawing to high extension ratios of melt-extruded VGCF-iPP nanocomposite filaments results in remarkable increases in modulus and strength, which mask any contribution of nano-reinforcement or inter-particle confinement observed previously. Based on X-ray diffraction and thermal analyses it is shown that the drawing process involves crumpling of the original 'shish-kebab' structure, accompanied by lateral shrinkage of the 'kebabs', disappearance and reorientation of daughter lamellae and significant increases in the degree of crystallinity by solid-state recrystallization. The effects of the initial diameter of the as-extruded filaments and those of the nanofibers and the 'shish-kebab' structure are dominated by the drawing process, which produces a highly aligned fibrillar structure with superior mechanical properties. A cartoon model of the effect of solid-state drawing of extruded iPP and nanocomposite filaments on the transition from 'shish-kebab' to fibril morphology is presented.