Oral Presentation 23rd International Society of Magnetic Resonance Conference 2023

Liquid-to-solid phase transition studied by solid-state NMR spectroscopy (#156)

Ettore Bartalucci 1 2 , Yaning Han 3 , Leonidas Emmanouilidis 3 , Wojciech Lipińksi 4 , Johannes Zehnder 5 , Evan Spruijt 4 , Frédéric Allain 3 , Thomas Wiegand 1 2
  1. Max Planck Institute for Chemical Energy Conversion, Mulheim an der Ruhr, NRW , Germany
  2. Institute of Technical and Macromolecular Chemistry, RWTH Aachen, Aachen, NRW, Germany
  3. Department of Biology, ETH Zurich, Zurich, Switzerland
  4. Institute for Molecules and Materials, Radboud University, Nijmegen, The Netherlands
  5. Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins or protein regions plays a pivotal role in a variety of biological processes and human diseases.[1,2] Insights into the liquid condensate formation via LLPS and the subsequent liquid-to-solid (LST) phase transition are therefore crucial for understanding the mechanisms of pathological aggregation into fibrillar amyloid-type structures, a phenomenon that has been associated with a variety of neurodegenerative diseases.[3] However, despite its importance, a molecular picture of the LST process inside the phase-separated droplets is still missing.

This contribution reports on a comprehensive investigation of liquid-droplet maturation processes by time-resolved solid-state NMR spectroscopy, in combination with solution-state NMR, and light and electron microscopies. Short peptide derivatives undergoing both LLPS and subsequent LST are discussed as model systems. Amyloid-like fibrils are formed upon LST and allow us establishing proof-of-concept methods.[4] In particular, a comparison across the condensed state for various constructs of the peptide derivatives highlights the important role of noncovalent interactions in the LST processes.

The discussion is then extended to LLPS phenomena of the RNA-binding protein Fused in Sarcoma (FUS), for which aberrant phase transition can lead to the formation of pathological aggregates. Such aggregates are considered as hallmarks of neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD).[5] The kinetics of FUS liquid droplets maturation into solid fibril species can be followed by real-time solid-state NMR, namely by detecting immobilized as well as highly mobile components with cross-polarization (CP) and Insensitive Nuclei Enhancement by Polarization Transfer (INEPT) experiments, respectively. Furthermore, two- and three-dimensional solid-state NMR experiments were recorded to characterize the molecular structure of the matured solid material formed during LST. The study sets the stage for characterizing the elusive transformations in structure and dynamics of proteins undergoing phase transitions from the liquid-droplet to the final fibrillar aggregate state.

  1. [1] Banani, S. F., et al. (2017). "Biomolecular condensates: organizers of cellular biochemistry." Nature reviews Molecular cell biology 18(5): 285-298.
  2. [2] Wang, B., et al. (2021). "Liquid–liquid phase separation in human health and diseases." Signal Transduction and Targeted Therapy 6(1): 1-16.
  3. [3] Ross, C. A. and M. A. Poirier (2004). "Protein aggregation and neurodegenerative disease." Nature medicine 10(7): S10-S17.
  4. [4] Lipiński, W. P., et al. (2022). "Fibrils emerging from droplets: Molecular guiding principles behind phase transitions of short peptide-based condensates." ChemRxiv.
  5. [5] Kato, M., et al. (2012). "Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels." Cell 149(4): 753-767