Tailoring solid-state DNP methods to the study of α-synuclein LLPS.

Area of Science:

• Biophysics
• Chemical Physics
• Spectroscopy

Background:

• Dynamic Nuclear Polarization (DNP) enhances Nuclear Magnetic Resonance (NMR) sensitivity using electron spins, crucial for analyzing challenging samples.
• Glassing agents are vital in DNP for polarization transfer and cryoprotection; glycerol-d8/D2O/H2O (DNPjuice) has been the standard for two decades.
• Polyethylene glycol (PEG) serves as a cryoprotectant and crowding agent, mimicking cellular environments for in vitro liquid-liquid phase separated (LLPS) condensates.

Purpose of the Study:

• To evaluate Polyethylene glycol (PEG) as a substitute for glycerol in DNP juice for enhanced signal detection.
• To explore the potential of PEG as a dual-function agent for both cryoprotection and inducing crowding in LLPS studies.

Main Methods:

• Investigated the efficacy of a PEG-water mixture as a glassing agent in Dynamic Nuclear Polarization (DNP) experiments.
• Utilized ASYMPOL-POK as a polarizing agent for cross-effect DNP enhancement on 13C-glycine without deuteration.
• Compared DNP enhancement and buildup rates with the traditional DNPjuice (glycerol-d8/D2O/H2O).

Main Results:

• The PEG-water matrix achieved high DNP enhancement, enabling direct solid-state DNP studies of LLPS condensates.
• PEG signals at ~72.5 ppm are well-separated from protein aliphatic regions, simplifying spectral analysis.
• DNP enhancement and buildup rates using PEG were comparable to those obtained with DNPjuice.

Conclusions:

• Polyethylene glycol (PEG) is an effective alternative to glycerol for DNP juice, functioning as both a glassing and crowding agent.
• The use of PEG facilitates direct solid-state DNP investigation of LLPS condensates without additional components.
• PEG offers a viable and advantageous matrix for DNP studies, particularly in mimicking cellular conditions and simplifying spectral interpretation.