Driving Force Of A-synuclein’s Liquid-liquid Phase Separation

α-Synuclein is a kind of protein related to Parkinson’s disease,which can aggregate into toxic aggregates in the brain,damaging the neuron and leading to the disease.Liquid–liquid phase separation（LLPS）,simply called phase separation,describes a biophysics phenomenon that biomolecules such as proteins assemble into micrometer-sized spherical condensates in specific solution environment.Research has shown thatα-Syn can phase separate and it’s phase separation promotes the aggregation.The target of this study is illustrating the driving force of LLPS ofα-Syn and finding out the segment that drives the LLPS of entire peptide.α-Syn and other two proteins（β-Syn andγ-Syn）of synuclein family were expressed with E.coli in this research and results of phase separation experiment suggested thatα-Syn as well asβ-Syn can phase separate forming droplets at the presence of high PEG8000 concentration,butγ-Syn not.Other studies have proposed that the central hydrophobic segment ofα-Syn plays an important role during the process of aggregation and inferred it’s also crucial in LLPS.Based on the fragment division and sequences alignment,we designed five truncation variants（P1-P5）ofα-Syn and another four variants（P6-P9）by substituting segments and expressed seven of them with E.coli.We screened out a good-use buffer in favor of LLPS usingα-Syn as model protein,and investigated the effects of salts and 1,6-hexanediol on LLPS ofα-Syn.We found that high concentration salt inhibits the occurrence of LLPS while hexanediol has little effect,andβ-Syn as well as P3has a similar result.These results suggest that electrostatic interaction between protein molecules is a significant role in LLPS but hydrophobic interaction not.By observing the LLPS of three nature proteins and seven designed proteins at different p H with a microscope we found that proteins containing the C terminal ofα-Syn or similar sequence are able to phase separate and form droplets,but others form amorphous precipitation or nothing.From this we concluded that the LLPS ofα-Syn andβ-Syn is driven by the electrostatic interaction of its C terminal.To verify the driving ability for LLPS of the C terminals ofα-Syn andβ-Syn,we substituted the C terminal ofγ-Syn withα-Syn’s andβ-Syn’s that can demix by themselves thus got two proteins PC and PD.To our surprise,we have not observed droplets formed by PC or PD,but amorphous precipitates or aggregates.By aligning the sequences ofα-Syn and PC we found the conservation score given by alignment program is lower at the 31^{t h},68^th,84^th and 86^th amino acid sites than those at other sites.Glycine residues occupy the four sites mentioned above inα-Syn,but in PC they are glutamic acid resiudes.Therefore we carried out a series of mutation experiments and demonstrated that LLPS ofα-Syn is not only governed by its C terminal but also affected by other amino acids.To sum up,this research illustrated the mechanism ofα-Syn’s LLPS from the perspective of sequence,and we hope to provide an idea for studying aggregation and LLPS ofα-Syn and expect the neurodegenerative disease including Parkinson’s disease could be cured at an early day.