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−15N 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.