Measuring long-range contacts in a fully protonated protein at 105 kHz magic angle spinning

Area of Science:

• Biomolecular solid-state Nuclear Magnetic Resonance (NMR) spectroscopy.
• Protein structure determination and characterization.
• Advanced NMR pulse sequence development.

Background:

• Proton (¹H) detection via fast magic-angle spinning (MAS) has advanced biomolecular NMR.
• Traditionally, deuteration was necessary for high-resolution protein structural analysis.
• Modern MAS rates (>100 kHz) reduce the need for deuteration, enabling studies of fully protonated systems.

Purpose of the Study:

• To evaluate the utility of 3D pulse sequences ((H)NCOH and (H)NCAH) for measuring long-range C-H correlations in fully protonated proteins.
• To assess the impact of high MAS rates on spectral quality and distance measurements.
• To determine the feasibility of NMR studies without deuteration and back-exchange.

Main Methods:

• Utilized two 3D pulse sequences: (H)NCOH and (H)NCAH.
• Performed experiments on a fully protonated protein sample.
• Employed a magic-angle spinning (MAS) rate of 105 kHz.
• Analyzed the resulting NMR spectra for long-range C-H correlations.

Main Results:

• The (H)NCOH spectrum revealed multiple sequential and structurally relevant long-range carbonyl-proton (CO-H) contacts per residue, up to 6 Å.
• (H)NCAH spectra showed fewer correlations, primarily intraresidue aliphatic proton contacts.
• Protonated systems exhibited dipolar truncation in Cα-H experiments, while CO-H correlations remained comparable to deuterated samples.

Conclusions:

• The (H)NCOH sequence is effective for measuring long-range CO-H distances in fully protonated proteins at high MAS rates.
• This approach facilitates distance measurements using long-range cross-polarization in more accessible and affordable samples.
• Expands the scope of proton detection NMR for systems where deuteration is not feasible.