Dynamics of lysine side-chain amino groups in a protein studied by heteronuclear ¹H-¹⁵N NMR spectroscopy

Despite their importance in macromolecular interactions and functions, the dynamics of lysine side-chain amino groups in proteins are not well understood. In this study, we have developed the methodology for the investigations of the dynamics of lysine NH₃⁺ groups by NMR spectroscopy and computation. By using ¹H-¹⁵ N heteronuclear correlation experiments optimized for ¹⁵NH₃⁺ moieties, we have analyzed the dynamic behavior of individual lysine NH₃ ⁺ groups in human ubiquitin at 2 °C and pH 5. We modified the theoretical framework developed previously for CH₃ groups and used it to analyze ¹⁵N relaxation data for the NH₃⁺ groups. For six lysine NH₃⁺ groups out of seven in ubiquitin, we have determined model-free order parameters, correlation times for bond rotation, and reorientation of the symmetry axis occurring on a pico-to nanosecond time scale. From CPMG relaxation dispersion experiment for lysine NH₃⁺ groups, slower dynamics occurring on a millisecond time scale have also been detected for Lys27. The NH₃⁺ groups of Lys48, which plays a key role as the linkage site in ubiquitination for proteasomal degradation, was found to be highly mobile with the lowest order parameter among the six NH₃⁺ groups analyzed by NMR. We compared the experimental order parameters for the lysine NH₃ ⁺ groups with those from a 1 μs molecular dynamics simulation in explicit solvent and found good agreement between the two. Furthermore, both the computer simulation and the experimental correlation times for the bond rotations of NH₃⁺ groups suggest that their hydrogen bonding is highly dynamic with a subnanosecond lifetime. This study demonstrates the utility of combining NMR experiment and simulation for an in-depth characterization of the dynamics of these functionally most important side-chains of ubiquitin.