Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation   

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Original research article

A brief version of this protocol appeared in:
Molecular Cell
Mar 2017


Studying liquid-liquid phase separation (LLPS) of proteins provides key insights into the biogenesis of membraneless organelles and pathological protein aggregation in disease. We have established a protocol for inducing the phase separation of arginine-rich peptides, which allows for studying their molecular determinants and dynamics (Boeynaems et al., 2017).

Keywords: LLPS, Amyotrophic lateral sclerosis, Peptides, Arginine, Phase separation, Mass spec, C9orf72, Stress granule


Arginine-rich disordered domains are often found in RNA binding proteins, including the ones associated with neurodegenerative diseases (e.g., FUS, FMRP, hnRNPA1) (Varadi et al., 2015; Boeynaems et al., 2017). Also toxic arginine-rich repeat peptides (i.e., polyGR and polyPR) are produced in amyotrophic lateral sclerosis patients carrying the C9orf72 repeat expansion (Kwon et al., 2014; Mizielinska et al., 2014; Varadi et al., 2015; Boeynaems et al., 2016). While phase separation of uncharged low complexity domains had been studied before (Kato et al., 2012; Burke et al., 2015; Lin et al., 2015; Molliex et al., 2015; Patel et al., 2015), we developed this protocol to test whether arginine-rich domains could also contribute to phase separation. A schematic representation of the protocol can be seen in Figure 1.

Figure 1. Workflow of protein droplet formation and analysis. A diffuse arginine-rich peptide solution can be induced to undergo liquid-liquid phase separation by addition of RNA or molecular crowder (PEG). Resulting droplets can be further analyzed by fluorescence microscopy and FRAP.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Boeynaems, S., De Decker, M., Tompa, P. and Van Den Bosch, L. (2017). Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation. Bio-protocol 7(17): e2525. DOI: 10.21769/BioProtoc.2525.

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