During the last decade, proteins that self-assemble into fluid membraneless organelles have attracted great attention in cellular biology. Such biomolecular condensates have been linked to fundamental functions and disease in vivo, and often emerge via liquid-liquid phase separation. Such phases can often be reproduced in vitro, but structural investigations remain very challenging. Their material properties – not an ordered solid, not freely tumbling molecules – and their conformational heterogeneity and dynamics at the microscopic level severely limit the reach of methods like solution NMR and cryo-electron microscopy. Magic Angle Spinning NMR applied to frozen samples has the potential to overcome some of those limitations, enabling the study of condensed phases of full length proteins.
Our model system is human FUS, a foremost example of a functionally relevant phase separating protein with direct implications in neurodegenerative diseases like Amyotrophic Lateral Sclerosis (Patel et al. 2015). FUS is a 526 amino acid long protein, mostly intrinsically disordered with very low sequence complexity. Transient interactions between the 24 Tyr residues in the N terminal prion-like domain, and the 26 Arg clustering in three so-called RGG domains are thought to be the one of the main driving forces in liquid-liquid phase separation, but a quantitative description of such contacts is missing.
By studying full length FUS samples at room temperature, but also after rapid freezing, we aim at the characterization of the condensed liquid phases, both accessing the underlying conformational ensemble and its dynamics. Our collaborator Massimiliano Bonomi at Institute Pasteur brings in cutting edge Molecular Dynamics methodologies that allow us to build a multiscale representation of the ensemble, and ultimately the condensed phase, using sparse experimental information. The Alberti group at TU Dresden provides us with expertise in the macroscopic characterization of the liquid phases and connects our research with results in cellular biology.
Reference: Patel A, Lee HO, Jawerth L, Maharana S, Jahnel M, Hein MY, Stoynov S, Mahamid J, Saha S, Franzmann TM, Pozniakovski A, Poser I, Maghelli N, Royer LA, Weigert M, Myers EW, Grill S, Drechsel D, Hyman AA, Alberti S. (2015) A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell 162(5):1066-77. DOI: 10.1016/j.cell.2015.07.047