Intrinsically disordered proteins/regions (IDPs/IDRs) represent one of biology's most intriguing puzzles with profound implications for human health. On one hand, the misfolding or aggregation of IDPs/IDRs is strongly linked to the development of neurodegenerative diseases; on the other hand, IDPs/IDRs can undergo a process called liquid-liquid phase separation (LLPS) to create membraneless organelles (MLOs), which allows biochemical reactions to occur in a highly organized and efficient manner within the crowded environment of the cell. Therefore, understanding the critical early steps of cellular organization — from the initial formation of protein oligomers before LLPS occurs, to how these small clusters evolve into larger, functional structures — ultimately reveals the essential role these dynamic processes play in maintaining life itself.
The challenge lies in the fact that nature doesn't always follow our conventional theories. Both theoretical and experimental evidence suggest that the early stages of LLPS may involve a multi-step process that differs from the traditional understanding of Classical Nucleation Theory (CNT). This makes it important to track the journey from single molecules to oligomer formation, and finally to the evolution of dense phases spanning tens of nanometers.
Since Liquid-phase Transmission Electron Microscopy (LP-TEM) can provide in situ nanoscale structural and dynamic information of biomolecules in their native solution state with millisecond temporal resolution, it has emerged as a powerful tool for studying single-molecule dynamics. This technique, when combined with molecular dynamics (MD) simulations, enables real-time monitoring of conformational ensembles of IDPs/IDRs, intermolecular interaction processes, the formation of molecular-to-nanoscale assemblies, and the dynamic behavior of aggregates in solution. More importantly, these processes are not unique to liquid-liquid phase separation (LLPS). Similar phenomena and theoretical frameworks are being explored in diverse fields including protein crystallization, macromolecular assembly, small organic molecule crystallization, and nanoparticle research.
Recently, Prof. Huan Wang's group at Peking University employed LP-TEM to directly observe and record the dynamic processes occurring during the early stages of liquid-liquid phase separation (LLPS). The study benefited from the expertise of Prof. Yiqin Gao, a theoretical chemist from Peking University, and Yihao Niu in molecular dynamics simulations, which provided deeper insights into the conformational ensembles of IDPs/IDRs. The findings, with Jiaye Li as the first author, were published in National Science Review under the title "Visualization of oligomerization, clustering, and density transition of intrinsically disordered proteins."This study elucidates the initial oligomerization of intrinsically disordered proteins/regions (IDPs/IDRs) through intermolecular interactions, the subsequent growth of these oligomers, and their eventual transition to a dense phase. By visualizing the evolution of oligomers into the dense phase, the study provides compelling evidence supporting a non-classical nucleation mechanism and proposes a multi-step process for the early stages of liquid-liquid phase separation (LLPS). Specifically, the low-complexity domain of the Fused in Sarcoma protein (FUS-LCD) was encapsulated within a graphene liquid cell (GLC), enabling direct observation of this multi-step LLPS process. The research focused on the formation of small oligomers from individual molecules and their subsequent clustering into a dense phase. LP-TEM experiments captured the dynamic formation of oligomers, revealing the presence of clusters even in undersaturated solutions. Quantitative image analysis further allowed the determination of the critical concentration for the dense phase transition and the driving force underlying this transition.
Similar transitions and dynamic processes were also observed in other IDPs/IDRs, including full-length Fused in Sarcoma protein (FL-FUS) and microtubule-associated protein Tau. This suggests that the dense phase transition is a common process in the interaction and LLPS of IDPs/IDRs. This provides direct experimental evidence for a non-classical, multi-step mechanism in the early stages of LLPS.
National Science Review
Imaging analysis