Deuterium Spin Probes of Backbone Order in Proteins: 2H NMR Relaxation Study of Deuterated Carbon α Sites
Devon Sheppard‡, Da-Wei Li§, Rafael Brschweiler§ and Vitali Tugarinov*‡
2H spin relaxation NMR experiments to study the dynamics of deuterated backbone α-positions, Dα, are developed. To date, solution-state 2H relaxation measurements in proteins have been confined to side-chain deuterons—primarily 13CH2D or 13CHD2 methyl groups. It is shown that quantification of 2H relaxation rates at Dα backbone positions and the derivation of associated order parameters of Cα−Dα bond vector motions in small [U-15N,13C,2H]-labeled proteins is feasible with reasonable accuracy. The utility of the developed methodology is demonstrated on a pair of proteins—ubiquitin (8.5 kDa) at 10, 27, and 40 °C, and a variant of GB1 (6.5 kDa) at 22 °C. In both proteins, the Dα-derived parameters of the global rotational diffusion tensor are in good agreement with those obtained from 15N relaxation rates. Semiquantitative solution-state NMR measurements yield an average value of the quadrupolar coupling constant, QCC, for Dα sites in proteins equal to 174 kHz. Using a uniform value of QCC for all Dα sites, we show that Cα−Dα bond vectors are motionally distinct from the backbone amide N−H bond vectors, with 2H-derived squared order parameters of Cα−Dα bond vector motions, S2CαDα, on average slightly higher than their N−H amides counterparts, S2NH. For ubiquitin, the 2H-derived backbone mobility compares well with that found in a 1-μs molecular dynamics simulation.