Periodic Reporting for period 3 - LLPS-NMR (Nuclear magnetic resonance spectroscopy of liquid-liquid phase separation)
Reporting period: 2021-09-01 to 2023-02-28
Liquid demixing is a phenomenon inherent to the thermodynamics of liquids, is critical for the development of technologically useful fluids and underlies some of the biggest health changes in our society. Despite the importance of LLPS in chemistry and biology, only a very low-resolution view primarily through light microscopy is currently available for LLPS states formed by peptides and low complexity proteins. Because of this bottleneck, the interactions, which stabilize liquid droplets, and regulate their biogenesis, as well as a rationale for the biochemical function of LLPS, are largely unknown. To tackle this massive unmet need, the ERC advanced project LLPS-NMR focuses on the development of novel methods of NMR spectroscopy and team them up with mechanobiology to break the resolution barrier of polypeptide LLPS and push the description of the internal organization of liquid droplets from micrometer to sub-nanometer. Although highly challenging, the novel methods when successful promise to (i) disentangle the structure and kinetics of intrinsically disordered proteins in LLPS reaction chambers in space and time, (ii) unravel the nature of chemical reactions in liquid droplets, and (iii) decipher LLPS regulation by posttranslational modifications, nucleic acids and critical changes in cellular environment at atomic resolution. By breaking the resolution barrier of liquid-liquid phase separation and pushing the description of the internal organization of aqueous phase-separated states from micrometer to sub-nanometer resolution, LLPS-NMR aims to transform our knowledge about the chemistry of liquid phase-separated protein states, to provide critical guidance in the development of systems to encapsulate bioactive molecules and to develop better treatments for human diseases.
During the first 2 1/2 years we made major advances in reaching the overall aims of of the LLPS-NMR project. We developed novel multidisciplinary approaches to describe the structure and dynamics of intrinsically disordered proteins in liquid phase-separated states at high resolution. We identified lysine as a critical residue for LLPS that regulates liquid-liquid phase separation and biomolecular condensation. We also gained unprecedented insights into how post-translational modifications govern the formation of biomolecular condensates, as well as molecular processes occurring inside of liquid phase-separated states. A major advance was also our finding that liquid-liquid phase separation plays an important role in gene transcription both for biology and in pathophysiological including transcription and replication of the SARS-CoV-2 coronavirus.
Several of our results are beyond the state of the art including the developed methods to reveal at unprecedented resolution the internal motions in proteins that undergo liquid-liquid phase separation, the demonstration that acetylation of lysine side chains is a critical regulator for liquid phase separation and the demonstration that liquid-liquid phase separation is a key process in gene transcription and replication of the SARS-CoV-2 coronavirus.