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

Large proteins and dilute spin systems within a deuterated background are often characterized by long proton (H) spin-lattice relaxation times (T), which directly impacts the recycle delay and hence, the total experimental time. Dioxygen (O) is a well-known paramagnetic species whose short electronic spin-lattice relaxation time (7.5 ps) contributes to effective spin-lattice relaxation of high gamma nuclei. Oxygen's chemical potential and high diffusivity also allows it to access both the protein exterior and much of the (hydrophobic) interior of the protein. Consequently, at O partial pressures of ~ 10 bar, H and F spin-lattice relaxation rates (R) typically reach 3-5 Hz (versus rates of 0.7-1.0 Hz without oxygen) with comparable line-broadening in protein NMR spectra. Using fluoroacetate dehalogenase (FAcD) a soluble 35 kDa homodimeric enzyme, a nanodisc-stabilized G protein-coupled receptor (AR), and bovine serum albumin (BSA) as test cases, a 3-fold savings in time was achieved in acquiring H- N HSQC and F NMR spectra, after oxygenation at 9 bar for 24 h. Additional spin-diffusion effects are anticipated to contribute to uniform H spin-lattice relaxation for both solvent-exposed and buried protons, as demonstrated by T relaxation analysis of amides in N-labeled FAcD. Finally, we show that in protein samples dissolved oxygen pre-equilibrated at 9 bar (pO) is largely retained in solution at 20° C or lower, using a standard NMR tube for a period of 3-4 days, thus avoiding the use of specialized apparatus or high-pressure NMR tubes in the spectrometer. The convenience of being able to add or remove the quenching species, while avoiding any complex apparatus in the NMR experiment, makes this a practical tool for both F, H- C, and H- N NMR studies of proteins.