An NMR Insight into The Liquid-Liquid Phase Separation of FUS

• Autor(a) principal: O’Toole, Philip Guilherme Capelo
• Data de Publicação: 2020
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/112885
• topic: liquid-liquid phase separation; FUS; NMR; protein-protein interactions; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

The formation of two distinct liquid phases from a homogenous solution of solvent and polymer is termed liquid-liquid phase separation (LLPS). The LLPS phenomenon has been described for polymers and proteins. The LLPS of proteins is crucial for the formation of membraneless organelles, which play an important role in intracellular compartmentalization, and is inherently linked to protein fibrillation found in neurodegenerative diseases. The protein fused in sarcoma (FUS) is present in protein fibrils found in amyotrophic lateral sclerosis patients and has been extensively studied as a model protein for LLPS. The protein contains a prion-like domain, two folded RNA binding domains, the RNA recognition motif (RRM) and a zinc finger domain (ZnF) and three arginine-glycine-glycine rich regions (RGG). The main molecular drivers and interactions underlying the LLPS phenomenon of FUS have been previously described, particularly for the LC domain. However, the role of the different regions in this structurally complex protein remains to be fully understood. In this work, the full-length protein was divided into two distinct previously unstudied constructs, namely RGG1-RRM-RGG2and RGG2-ZnF-RGG3. The LLPS propensity was examined, together with the molecular and structural drivers underlying the LLPS phenomenon of each construct. These objectives were addressed by performing turbidity assays and nuclear magnetic resonance spectroscopy (NMR). Based on the turbidity assays, the RGG1-RRM-RGG2construct appears to undergo LLPS when isolated. The folded RRM may play a direct role under certain conditions and RNA binding significantly affects the observed LLPS propensity. Moreover, at least ionic, and π-cation interactions act as drivers for the LLPS phenomenon of this domain. Through NMR spectroscopy, several residues of the folded RRM domain were significantly affected by temperature, sample cooling and reheating procedures and protein concentration. The specifically affected residues are likely to participate in protein-protein interactions, therefore establishing a hypothetical protein-protein interaction in the folded RRM domain. Due to the structural localization of the interface, the protein-protein interactions established by the folded RRM domain are not directly affected by RNA-binding. The RGG2-ZnF-RGG3 construct, as observed through NMR spectroscopy, presented an apparent cluster of H-bond performing residues within the folded ZnF domain, namely in the vicinity of the residues coordinating the Zn2+ ion. Furthermore, the construct did not appear to undergo LLPS under the tested conditions. To fully certify the absence of LLPS in this construct, further assays must be performed under distinct conditions. A previously unstudied FUS based construct revealed the capacity to undergo LLPS, driven at least by ionic, π-cation interactions and with direct participation of the folded RRM domain. The contribution of the RGG regions on the observed LLPS phenomenon remains to be elucidated. The tested RGG2-ZnF-RGG3did not undergo LLPS under the tested conditions. Further assays are necessary to evaluate the LLPS capacity of the construct.