Pushing Sensitivity and Specificity Limits in Native Structural Biology: ¹⁹F Multinuclear Dynamic Nuclear...

Area of Science:

• Biochemistry
• Biophysics
• Structural Biology

Background:

• Understanding protein structure and interactions is crucial for cellular processes and drug development.
• Determining atomic-level protein structural changes in cellular environments remains a significant challenge.
• Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool for molecular structure determination.

Purpose of the Study:

• To develop a highly sensitive and specific NMR method for atomic-level protein structural analysis in mammalian cells.
• To overcome the limitations of traditional NMR in detecting proteins within complex cellular matrices.
• To establish a framework for investigating protein structure, dynamics, and interactions in vivo.

Main Methods:

• Development of a fluorine-19 (19F)-based, proton (1H)-assisted dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR technique.
• Application of 1H-19F cross-polarization (CP) for enhanced sensitivity and 19F-13C double CP for structural information.
• Utilizing 1H-19F-13C magnetization transfer for selective detection of protein residues near a fluorine label.

Main Results:

• Achieved background-free detection of target proteins in mammalian cells with exceptional sensitivity and specificity.
• Demonstrated the methodology using human Cyclophilin A (CypA) in A2780 cells, incorporating a single fluorine atom.
• Successfully detected 13C signals from CypA residues up to 6 Å away from the fluorine label via magnetization transfer.

Conclusions:

• The developed 19F DNP MAS NMR approach provides a sensitive and specific tool for atomic-level protein structural analysis in mammalian cells.
• This methodology enables the investigation of protein structure, dynamics, and interactions within their native cellular context.
• The study establishes a robust framework for advancing in vivo protein studies using NMR spectroscopy.