NMR Spectroscopy of Biomolecular Liquid-Liquid Phase Separation
von Crhistian Felipe Pantoja Rivillas
Datum der mündl. Prüfung:2023-03-10
Erschienen:2024-02-09
Betreuer:Prof. Dr. Markus Zweckstetter
Gutachter:Prof. Dr. Marina Bennati
Gutachter:Prof. Dr. Stefan Jakobs
Dateien
Name:Thesis_cpantoj_2024_M5.pdf
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Description:Main Document
Zusammenfassung
Englisch
Phase separation of biomolecules has transformed our understanding of cell organization and function, motivating intensive research in the last decade. New evidence for their critical role in mechanisms essential for cellular function has been uncovered. However, little is known regarding the molecular factors that govern their function, and techniques capable of probing their intrinsically dynamic nature are required. Nuclear magnetic resonance (NMR) spectroscopy is a powerful method to study biomolecules in solution, with its flexibility making it very attractive to study transitory and dynamic systems. In manuscript 1, we first explore the kinetics of droplet formation using NMR spectroscopy on a model system. The simplicity and versatility of the triethylamine (TEA)/water system offers a detailed description of the nucleation process and demixing kinetics. Notably, we detected a chemical exchange process between nucleation sites and matrix solution in the slow time scale of NMR. The corresponding exchange rate was quantified using exchange spectroscopy (EXSY) mediated by cooling/heating cycling. Finally, we illustrated the potential of our strategy by quantifying the exchange rate of a client molecule in the system. In manuscript 2, we analyze the potential of NMR to study non-uniform samples or two-phase systems. We developed an improved spatially resolved NMR experiment to study a phase-separated sample of Tau protein. By including a suitable water suppression block, multicomponent quantification of small and large molecules was feasible. We provide an extended strategy to quantify protein concentration in a spatially resolved fashion. Additionally, we presented a quantitative phase diagram of Tau protein using spatially resolved NMR. Finally, in manuscript 3 we demonstrate the application of NMR to study biochemical reactions inside biomolecular condensates. Here, we monitored in real-time the phosphorylation process of Tau condensate mediated by the kinase CDK2. The kinetic data revealed that phosphorylation inside Tau condensate is accelerated compared to dispersed phase. Altogether, the present findings push the borders of the applicability of NMR to challenging phase separate systems, extending our understanding of the molecular factors behind the phase separation process.
Keywords: Biomolecular condesates; NMR spectroscopy; Spatially resolved NMR; Exchange spectroscopy