Real-time observation of structure and dynamics during the liquid-to-solid transition of FUS LC

A subset of the proteins found in pathological protein fibrils also exhibit tendencies for liquid-liquid phase separation (LLPS) both in vitro and in cells. The mechanisms underlying the connection between these phase transitions have been challenging to study due to the heterogeneous and dynamic nature of the states formed during the maturation of LLPS protein droplets into gels and solid aggregates. Here, we interrogate the liquid-to-solid transition of the low-complexity domain of the RNA-binding protein FUS (FUS LC), which has been shown to adopt LLPS, gel-like, and amyloid states. We employ magic-angle-spinning NMR spectroscopy, which has allowed us to follow these transitions in real time and with residue-specific resolution. We observe the development of beta-sheet structure through the maturation process and show that the final state of FUS LC fibrils produced after LLPS is distinct from that grown from fibrillar seeds. We also apply our methodology to FUS LC G156E, a clinically relevant FUS mutant that exhibits accelerated fibrillization rates. We observe significant changes in dynamics during the transformation of the FUS LC G156E construct and begin to unravel the sequence specific contributions to this phenomenon with computational studies of the phase-separated state of FUS LC and FUS LC G156E. SIGNIFICANCE The presence of protein aggregates in the brain is a common pathological sign of neurodegenerative disease. Recent work has revealed that a subset of the proteins found in these aggregates can also form liquid-liquid droplets and gels. Although the interconversion from one state to another can have vast implications for cell function and disease, the molecular mechanisms that underlie these processes are not well understood. Here, we combine magicangle-spinning NMR spectroscopy with other biophysical and computational tools to follow the transitions of the RNAbinding protein FUS. This approach has allowed us to observe real-time changes in structure and dynamics as the protein undergoes these transitions, and to reveal the intricate effects of disease-relevant mutations on the transformation process.

Automated summary

Proteins found in pathological fibrils can exhibit liquid-liquid phase separation tendencies, with real-time tracking revealing structural transitions in RNA-binding protein FUS. Disease-relevant mutations have significant effects on the process.