Deuterium spin probes of backbone order in proteins: 2H NMR relaxation study of deuterated carbon alpha sites.

(2)H spin relaxation NMR experiments to study the dynamics of deuterated backbone alpha-positions, D(alpha), are developed. To date, solution-state (2)H relaxation measurements in proteins have been confined to side-chain deuterons-primarily (13)CH(2)D or (13)CHD(2) methyl groups. It is shown that quantification of (2)H relaxation rates at D(alpha) backbone positions and the derivation of associated order parameters of C(alpha)-D(alpha) bond vector motions in small [U-(15)N,(13)C,(2)H]-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 degrees C, and a variant of GB1 (6.5 kDa) at 22 degrees C. In both proteins, the D(alpha)-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(alpha) sites in proteins equal to 174 kHz. Using a uniform value of QCC for all D(alpha) sites, we show that C(alpha)-D(alpha) bond vectors are motionally distinct from the backbone amide N-H bond vectors, with (2)H-derived squared order parameters of C(alpha)-D(alpha) bond vector motions, S(2)(CalphaDalpha), on average slightly higher than their N-H amides counterparts, S(2)(NH). For ubiquitin, the (2)H-derived backbone mobility compares well with that found in a 1-mus molecular dynamics simulation.