Carbon relaxation in 13Cα-Hα and 13Cα-Dα spin pairs as a probe of backbone dynamics in proteins.

NMR methodology for the measurements of α-carbon R(1) and R(1ρ) spin relaxation rates in (13)C(α)-H(α) and (13)C(α)-D(α) spin pairs of U-[(13)C; (15)N] partially deuterated proteins is developed. The intra-HN[CA] NMR experiment isolates carbon nuclei belonging to either (13)C(α)-H(α) or (13)C(α)-D(α) spin systems in the same protein sample prior to the measurement of (13)C(α) relaxation rates. The differences between R(1) and R(2) rates in the two spin pairs (ΔR(1), ΔR(2)) eliminate all contributions to (13)C(α) decay rates not associated with direct (13)C(α)-(1)H(α)(D(α)) dipolar interactions including chemical exchange and serve as robust measures of C(α)-H(α)(D(α)) bond vector motions in proteins. The methodology is applied to the relaxation study of α-carbon sites in the protein ubiquitin at two temperatures. The measures of order of individual C(α)-H(α)(D(α)) bond vectors (S(2)) in ubiquitin derived from the fitting of differential rates (ΔR(1), ΔR(2)) unambiguously report on protein dynamics, thereby eliminating potential contributions from modulations of C(α)-H(α)(D(α)) bond lengths by their environment. They are comparable to the ones obtained from a molecular dynamics simulation at 27 °C.