Dynamics of Lysine Side-Chain Amino Groups in a Protein Studied by Heteronuclear ¹H−¹⁵N NMR Spectroscopy

Abstract

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.

Signature of Mobile Hydrogen Bonding of Lysine Side Chains from Long-Range 15N–13C Scalar J-Couplings and Computation

“…5 For ubiquitin, the majority of the lysine NH 3 þ groups display notably low S 2 order parameters, reflecting a high degree of internal mobility for these functionally important entities, with correlation times in the subnanosecond range for the NH 3 þ bond rotations. However, the origin of the side-chain mobility could not be elucidated from experiment alone.…”

“…13 To ensure a fair comparison, the simulation was run at the same temperature as in the experiment; otherwise, the MD simulation protocol was the same as described previously. 5 The length of the MD simulation was expected to be adequate for this analysis, since our previous NMR relaxation study showed that the reorientation of the symmetry axes of lysine NH 3 þ groups in ubiquitin occurs on the subnanosecond time scale. 5 Values of AE 3 J NζCγ ae were calculated from 1000 MD snapshots sampled every 1 ns.…”

“…5 The length of the MD simulation was expected to be adequate for this analysis, since our previous NMR relaxation study showed that the reorientation of the symmetry axes of lysine NH 3 þ groups in ubiquitin occurs on the subnanosecond time scale. 5 Values of AE 3 J NζCγ ae were calculated from 1000 MD snapshots sampled every 1 ns. As shown in Figure 2C, the AE 3 J NζCγ ae data from the MD ensemble exhibited substantially improved agreement with the experimental 3 J NζCγ data (rmsd = 0.26 Hz).…”

Measurement and Applications of Long‐Range Heteronuclear Scalar Couplings: Recent Experimental and Theoretical Developments

“…[64] Use of short-and long-range 15 C/ 15 N correlation in the lysine side chain in ubiquitin. [21,65] One of them is the intra-residue 15 N z and 13 C g coupling while the other is between the 15 N z and carbonyl 13 C across a hydrogen bond, as shown in Figure 6 J NzCg values are also calculated from the experimental 3 J NzCg and torsional angles obtained from the crystal structure by employing the empirical Karplus equation.…”

“…[19] It also provides the signature of the hydrogen bond in either small or medium-sized molecules such as benzamide [20] and its related compounds or the side-chain residue of protein molecules, thereby giving access to dynamic information about the protein side chains. [21] It has also received considerable attention in recent times in establishing configurations of bigger molecules. [22] Herein, we mainly focus on recent experimental methods designed for the measurement of n J XY , with special emphasis on n J CH , their applications and recent theoretical developments.…”