Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP...

Area of Science:

• Solid-state Nuclear Magnetic Resonance (NMR) Spectroscopy
• Biomolecular Structure Determination
• Dynamic Nuclear Polarization (DNP)

Background:

• Dynamic Nuclear Polarization (DNP) enhances NMR signal sensitivity but faces challenges with spectral congestion and line broadening at cryogenic temperatures.
• Complex biomolecules and systems with chemical-shift degeneracy often present difficulties in standard NMR analysis.
• Accurate structural determination of biomolecules is crucial for understanding their function.

Purpose of the Study:

• To develop an experimental strategy that enhances the efficiency and reliability of DNP-enhanced solid-state NMR.
• To overcome spectral congestion and line broadening issues associated with cryogenic DNP conditions.
• To facilitate faster and more accurate de novo resonance assignments for complex biomolecules.

Main Methods:

• Integration of sequential correlation experiments for side-chain and Cα resonances.
• Implementation of Non-Uniform Sampling (NUS) for efficient multidimensional data acquisition.
• Utilization of fast (25 kHz) magic-angle spinning (MAS) to optimize spectral resolution and sensitivity.

Main Results:

• A virtually complete de novo assignment of the Pf1 virus coat protein was achieved in under one week.
• The developed method effectively addresses spectral congestion and line broadening at cryogenic temperatures.
• Temperature-dependent perturbations (100 K vs. 273 K) were observed and correlated with hydration surfaces.

Conclusions:

• The novel DNP strategy significantly improves the efficiency of solid-state NMR for complex biomolecules.
• This approach enables simpler, faster, and more reliable resonance assignments, even in challenging systems.
• The findings provide insights into temperature-induced spectral changes and their relation to biomolecular hydration.