Reducing experimental time through spin-lattice relaxation enhancement via dissolved oxygen

Area of Science:

• Biophysics
• Structural Biology
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• Long proton (¹H) spin-lattice relaxation times (T₁), common in large proteins and dilute spin systems, increase experimental duration in NMR.
• Dioxygen (O₂), a paramagnetic species, effectively enhances spin-lattice relaxation rates (R₁) for high gamma nuclei, including ¹H and ¹⁹F.

Purpose of the Study:

• To investigate the impact of dissolved oxygen on ¹H and ¹⁹F spin-lattice relaxation in various protein systems.
• To evaluate the potential for time savings in NMR experiments using oxygenation.
• To assess the stability and practical application of oxygenation in standard NMR setups.

Main Methods:

• Oxygenation of protein samples (fluoroacetate dehalogenase, A₂A receptor, bovine serum albumin) with O₂ at ~9-10 bar.
• Measurement of ¹H and ¹⁹F spin-lattice relaxation rates (R₁) before and after oxygenation.
• Acquisition of ¹H-¹⁵N HSQC and ¹⁹F NMR spectra to assess time savings.
• Analysis of T₁ relaxation and oxygen retention in standard NMR tubes.

Main Results:

• Oxygenation at ~9 bar increased ¹H and ¹⁹F R₁ rates to 3-5 Hz, compared to 0.7-1.0 Hz without oxygen.
• A threefold reduction in experimental time was achieved for ¹H-¹⁵N HSQC and ¹⁹F NMR spectra.
• Spin-diffusion effects contributed to uniform ¹H spin-lattice relaxation for both solvent-exposed and buried protons.
• Dissolved oxygen levels remained stable in standard NMR tubes at ≤20°C for 3-4 days.

Conclusions:

• Dissolved oxygen is a practical and effective agent for accelerating spin-lattice relaxation in protein NMR.
• This method offers significant time savings for ¹⁹F, ¹H-¹³C, and ¹H-¹⁵N NMR studies without requiring specialized equipment.
• The ability to easily introduce or remove oxygen makes it a versatile tool for optimizing NMR experiments.