Deuterium Spin Probes of Backbone Order in Proteins: ~2H NMR Relaxation Study of Deuterated Carbon a Sites

摘要

~2H spin relaxation NMR experiments to study the dynamics of deuterated backbone a-positions, D~α, are developed. To date, solution-state ~2H relaxation measurements in proteins have been confined to side-chain deuterons-primarily ~(13)CH_2D or ~(13)CHD_2 methyl groups. It is shown that quantification of ~2H relaxation rates at D~α backbone positions and the derivation of associated order parameters of C~α-D~α bond vector motions in small [U-~(15)N,~(13)C,~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 ℃, and a variant of GB1 (6.5 kDa) at 22 ℃. In both proteins, the D~α-derived parameters of the global rotational diffusion tensor are in good agreement with those obtained from ~(15)N relaxation rates. Semiquantitative solution-state NMR measurements yield an average value of the quadrupolar coupling constant, QCC, for D~α sites in proteins equal to 174 kHz. Using a uniform value of QCC for all D~α sites, we show that C~α-D~α bond vectors are motionally distinct from the backbone amide N-H bond vectors, with 2H-derived squared order parameters of C~α-D~α bond vector motions, S~_(CαDα), on average slightly higher than their N-H amides counterparts, S~2_(NH). For ubiquitin, the ~2H-derived backbone mobility compares well with that found in a 1-μs molecular dynamics simulation.