Deuterium spin relaxation of fractionally deuterated ribonuclease H using paired 475 and 950 MHz NMR spectrometers.

Deuterium (²H) spin relaxation of ¹³CH₂D methyl groups has been widely applied to investigate picosecond-to-nanosecond conformational dynamics in proteins by solution-state NMR spectroscopy. The B₀ dependence of the ²H spin relaxation rates is represented by a linear relationship between the spectral density function at three discrete frequencies J(0), J(ω_D) and J(2ω_D). In this study, the linear relation between ²H relaxation rates at B₀ fields separated by a factor of two and the interpolation of rates at intermediate frequencies are combined for a more robust approach for spectral density mapping. The general usefulness of the approach is demonstrated on a fractionally deuterated (55%) and alternate ¹³C-¹²C labeled sample of E. coli RNase H. Deuterium relaxation rate constants (R₁, R_1ρ, R_Q, R_AP) were measured for 57 well-resolved ¹³CH₂D moieties in RNase H at ¹H frequencies of 475 MHz, 500 MHz, 900 MHz, and 950 MHz. The spectral density mapping of the 475/950 MHz data combination was performed independently and jointly to validate the expected relationship between data recorded at B₀ fields separated by a factor of two. The final analysis was performed by jointly analyzing 475/950 MHz rates with 700 MHz rates interpolated from 500/900 MHz data to yield six J(ω_D) values for each methyl peak. The J(ω) profile for each peak was fit to the original (τ_M, S_f², τ_f) or extended model-free function (τ_M, S_f², S_s², τ_f, τ_s) to obtain optimized dynamic parameters.