Multiplexed Analysis of Multicomponent Biomolecular Condensates without Any Tag.

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Biomolecular condensates are crucial for cellular organization and RNA metabolism, forming via liquid-liquid phase separation (LLPS).
• Current experimental methods often use fluorophore labeling, which can alter condensate behavior and prevent studying molecules in their native chemical state.

Purpose of the Study:

• To introduce a noninvasive, multiplexed Nuclear Magnetic Resonance (NMR) approach for studying biomolecular condensates.
• To enable the selective observation of multiple components within condensates without the need for chemical tags or labels.

Main Methods:

• Development of a multiplexing filter combining enhanced spin-spin cross-relaxation, diffusion, and isotope filters.
• Application of a 1D 1H NMR experiment to biphasic condensate samples.
• Integration of the multiplexing filter into multidimensional NMR experiments.

Main Results:

• Selective observation of multiple protein components in hydrogel-stabilized biphasic condensates without tags.
• Demonstration of the method's robustness with FUS protein condensates and a multicomponent hnRNPC1 condensate.
• Successful resolution of signals from distinct protein pools in both dilute and condensed phases.

Conclusions:

• The developed multiplexed NMR approach provides a noninvasive method to study biomolecular condensates.
• This technique allows for site-specific structural and dynamic insights into diverse protein populations within condensates.
• The method significantly extends the application of NMR spectroscopy in the field of LLPS research.