A high-field cellular DNP-supported solid-state NMR approach to study proteins with sub-cellular specificity

Area of Science:

• Biophysics
• Structural Biology
• Cellular Biology

Background:

• Studying protein structures in sub-cellular compartments is crucial.
• Dynamic nuclear polarization supported solid-state NMR (DNP-ssNMR) offers potential but suffers from low sensitivity and resolution.

Purpose of the Study:

• To improve DNP-ssNMR sensitivity and resolution for in-cell structural studies.
• To investigate the structural plasticity of proteins within specific cellular locations.

Main Methods:

• Utilized a novel biradical, SNAPol-1, for DNP-ssNMR experiments.
• Conducted experiments on [13C, 15N] labeled ubiquitin within intact HeLa cells and isolated cell nuclei.
• Employed high-magnetic fields (800 MHz/527 GHz).

Main Results:

• SNAPol-1 demonstrated homogeneous diffusion and distribution within whole cells and isolated nuclei.
• Achieved high sensitivity and significantly improved spectral resolution for ubiquitin.
• Physical enrichment of cell nuclei reduced measurement time by 4-fold, providing a unique view of nuclear ubiquitin.

Conclusions:

• SNAPol-1 enhances DNP-ssNMR for atomic-level protein structure analysis within sub-cellular compartments.
• This advancement allows for detailed investigation of protein conformational plasticity with sub-cellular specificity.
• The method opens new avenues for understanding protein function in its native cellular environment.