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

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

Abstract

Large proteins and dilute spin systems within a deuterated background are often characterized by long proton (1H) spin-lattice relaxation times (T1), which directly impacts the recycle delay and hence, the total experimental time. Dioxygen (O2) 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. Oxygenâ??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 O2 partial pressures of ~â??10 bar, 1H and 19F spin-lattice relaxation rates (R1) typically reach 3â??5Â*Hz (versus rates of 0.7-1.0Â*Hz without oxygen) with comparable line-broadening in protein NMR spectra. Using fluoroacetate dehalogenase (FAcD) a soluble 35Â*kDa homodimeric enzyme, a nanodisc-stabilized G protein-coupled receptor (A2AR), and bovine serum albumin (BSA) as test cases, a 3-fold savings in time was achieved in acquiring 1H-15Â*N HSQC and 19F NMR spectra, after oxygenation at 9Â*bar for 24Â*h. Additional spin-diffusion effects are anticipated to contribute to uniform 1H spin-lattice relaxation for both solvent-exposed and buried protons, as demonstrated by T1 relaxation analysis of amides in 15N-labeled FAcD. Finally, we show that in protein samples dissolved oxygen pre-equilibrated at 9Â*bar (pO2) is largely retained in solution at 20° C or lower, using a standard NMR tube for a period of 3â??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 19F, 1H-13Â*C, and 1H-15Â*N NMR studies of proteins.



Source: Journal of Biomolecular NMR