光谱学
荧光光谱法
荧光
荧光互相关光谱
荧光相关光谱
分析化学(期刊)
相(物质)
化学
材料科学
分离(统计)
色谱法
光学
物理
有机化学
计算机科学
机器学习
量子力学
作者
Wenxin Yu,Jian Liu,Xiangyi Huang,Jicun Ren
出处
期刊:Langmuir
[American Chemical Society]
日期:2023-12-29
标识
DOI:10.1021/acs.langmuir.3c02711
摘要
Liquid–liquid phase separation (LLPS) of fused in sarcoma (FUS) has emerged as a fundamental principle underpinning cellular function and malfunction. However, we know little about the FUS phase transition process from individual molecules to nanoscale condensates, which plays important roles in neurodegenerative diseases. Here, we propose the fluorescence correlation spectroscopy (FCS) method to quantitatively study the phase separation process of FUS protein with the fluorescent tag-enhanced green fluorescent protein (EGFP), from individual molecules to nanoscale condensates. The characteristic diffusion time (τD) of the protein condensates can be obtained from the FCS curve, which increases with the growth of the protein hydration radius. The bigger the τD value of the protein condensates, the larger the condensates formed by the phase separation of FUS. By this method, we discovered that the critical concentration for FUS to phase separation was 20 nM. We then plotted FUS phase diagrams based on τD under different concentrations of NaCl and found that both low-salt and high-salt concentrations tended to promote FUS-EGFP phase separation. Our results showed that ATP has a good inhibitory effect on FUS phase separation, and its inhibition constant IC50 was 3.2 mM. Finally, we evaluated the inhibition efficiency of single-stranded DNA sequences (ssDNA) on FUS phase separation and demonstrated that ssDNA containing three copies of TCCCCGT had relatively strong inhibition efficiency. In summary, our work provides detailed insight into the FUS phase transition process from individual molecules to nanoscale condensates at nanomolar concentrations and can be exploited for drug screening of neurodegenerative diseases.
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