Protein in-cell NMR spectroscopy at 1.2 GHz

Area of Science:

• Biophysical Chemistry
• Structural Biology
• Cellular NMR Spectroscopy

Background:

• Nuclear Magnetic Resonance (NMR) spectroscopy offers atomic-resolution insights into biomolecules within cells.
• Low sensitivity is a major limitation for in-cell NMR applications.
• Ultrahigh magnetic fields, like those in 1.2 GHz NMR, may negatively impact cell samples.

Purpose of the Study:

• To evaluate the performance of a 1.2 GHz NMR spectrometer for in-cell NMR on human cells.
• To compare resolution and sensitivity at 1.2 GHz against lower field strengths (900 and 950 MHz).
• To assess the impact of ultrahigh fields on cellular samples and protein dynamics.

Main Methods:

• Acquisition of in-cell NMR spectra at 1.2 GHz on human cells.
• Comparison of spectral data with spectra recorded at 900 and 950 MHz.
• Recording of SOFAST-HMQC and BEST-TROSY pulse sequences on intracellular alpha-synuclein and carbonic anhydrase.
• Evaluation of spin relaxation rates and their effect on spectral quality.

Main Results:

• The first in-cell NMR spectra at 1.2 GHz were successfully recorded on human cells.
• Significant improvements in resolution and sensitivity were observed at 1.2 GHz, particularly for unfolded proteins like alpha-synuclein.
• The TROSY (Transverse Relaxation-Optimized Spectroscopy) technique enhanced spectral resolution for both globular and unfolded proteins.
• Specific pulse sequences (SOFAST-HMQC, BEST-TROSY) were utilized to probe protein dynamics and relaxation behavior.

Conclusions:

• The 1.2 GHz NMR spectrometer significantly advances in-cell NMR capabilities, especially for studying intrinsically disordered proteins.
• The TROSY method is crucial for improving spectral quality in cellular environments at ultrahigh fields.
• This work validates the potential of ultrahigh-field in-cell NMR for detailed structural and functional studies of biomacromolecules in their native state.