Conformations of a Low-Complexity Protein in Homogeneous and Phase-Separated Frozen Solutions.

Area of Science:

• Biophysics
• Protein Science
• Molecular Biology

Background:

• Liquid-liquid phase separation (LLPS) is crucial in biological processes and protein biophysics.
• Intrinsically disordered proteins, like FUS-LC, undergo LLPS, raising questions about conformational changes in dense phases.
• High concentrations and interactions within phase-separated droplets may alter protein conformations.

Purpose of the Study:

• To investigate if local conformational distributions differ between homogeneous and phase-separated states of FUS-LC.
• To determine the extent of conformational changes accompanying LLPS in intrinsically disordered proteins.
• To assess the impact of LLPS on protein structure at a local level.

Main Methods:

• Solid-state nuclear magnetic resonance (ssNMR) spectroscopy was employed to analyze FUS-LC.
• Solutions were rapidly frozen after equilibration above and below the LLPS transition temperature.
• Dynamic nuclear polarization (DNP) enhanced ssNMR measurements were performed at low temperatures (25 K).

Main Results:

• Two-dimensional ssNMR spectra showed nearly identical crosspeak patterns for both homogeneous and phase-separated FUS-LC states.
• Labeling strategies (uniform, residue-specific, fragment ligation) yielded consistent results.
• Simulations indicated that conformational distribution changes are minimal (≤ 5-10%).

Conclusions:

• LLPS of FUS-LC does not induce substantial changes in local conformational distributions.
• The structural integrity of intrinsically disordered proteins is maintained across different phase states.
• ssNMR is a powerful tool for probing protein conformations in complex biological condensates.