Nuclear Magnetic Resonance Based Characterization of Liquid-Liquid Phase Separation

Liquid-liquid phase separation (LLPS) is increasingly recognized as an important phenomenon in cell biology and biotechnology. LLPS manifests itself as small dense droplets suspended in a dilute phase (micro LLPS), and once the droplets become large enough, a distinct dense and liquid-poor layer usually merges in the sample (macro LLPS). Layer separation usually represents the final stage of macroscopic LLPS. Scientists are very interested in the kinetics of these processes and the effect of different conditions or additives on these kinetics. However, the emulsion and concentration fluctuations make these processes difficult to study by current techniques. In addition, layer separation adds a complex spatial component because of the inhomogeneous phase distribution throughout the sample.

Customized Services

Our team of experts has extensive experience in the kinetic characterization of phase transitions and layer separations, and is committed to selecting advanced characterization techniques to achieve a more comprehensive assessment, especially regarding the evolution of the concentration and volume of the two phases. Here, CD BioSciences offers the powerful nuclear magnetic resonance (NMR) spectroscopy to characterize protein LLPS. Our high resolution multidimensional approach has been used to probe the molecular interactions between biological LLPS, as well as the separated thin and dense fractions. We can help you obtain the following information:

✓ Protein structural features.

✓ Conformational changes of intrinsically disordered proteins (IDPs) during phase separation. Based on the chemical shifts of ¹H, ¹³C, ¹⁵N, ¹⁹F and ³¹P nuclei, we help you to analyze the dynamic changes of protein backbone and side chains during LLPS.

✓ Diffusion coefficients. We provide pulsed field gradient (PFG) NMR to determine the diffusion coefficients of biomolecular condensates where the resonances of different species can be separated according to molecular size.

✓ Kinetic, thermodynamic and structural information. We offer nuclear spin relaxation (NSR) techniques to probe protein dynamics on ps to ns time scales, providing relevant information on bond vibrations/liberations, side chain rotational isomer reorientation and backbone torsion angle rotation. In addition, we have relaxation dispersion methods that provide kinetic, thermodynamic, and structural information on chemical exchange processes occurring on μs to ms time scales.

Proudly, facing the challenge that conventional high-resolution protein NMR cannot study the actual processes of phase separation, especially layer separation. We offer an approach that uses fluorinated probe molecules with bulk detection and spatially selective NMR analysis to fully characterize the entire processes and kinetics of micro- and macroscopic protein LLPS. The advantages of this strategy are:

• The small fluorinated probes interact transiently with the protein molecules, generating different NMR signals for each evolutionary phase.
• Each characteristic signal reflects the properties of each individual phase, in particular the phase volume and concentration.
• Both phases can be monitored and quantified simultaneously by their characteristic signals.

We offer conventional high-resolution NMR experiments that can be applied under idealized conditions prior to LLPS, either by suspending a small number of dense droplets in solution, or in isolated fractions. In addition we have spatially selective NMR capable of characterizing the spatial distribution of the two phases, especially during layer separation. If you have any special requirements for our services, please feel free to contact us. We are looking forward to working together with your attractive projects.