Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas 77555, United States.
J Am Chem Soc. 2011 Feb 2;133(4):909-19. doi: 10.1021/ja107847d.
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.
尽管赖氨酸侧链氨基在生物大分子相互作用和功能中具有重要意义，但人们对其动力学性质的了解还很有限。在本研究中，我们开发了利用 NMR 光谱学和计算方法研究赖氨酸 NH3+ 基团动力学的方法。通过使用针对 15NH3+ 部分优化的 1H-15N 异核相关实验，我们分析了 2°C 和 pH 5 下人类泛素中单个赖氨酸 NH3+ 基团的动态行为。我们对之前为 CH3 基团开发的理论框架进行了修改，并将其用于分析 NH3+ 基团的 15N 弛豫数据。在泛素中，除了一个赖氨酸 NH3+ 基团外，我们还确定了其余六个赖氨酸 NH3+ 基团的无模型主链构象参数、键旋转相关时间和对称轴重取向的相关时间，这些时间尺度在皮秒到纳秒之间。通过 CPMG 弛豫色散实验，我们还检测到 Lys27 上毫秒时间尺度上的较慢动力学。在泛素化和蛋白酶体降解的连接位点起关键作用的 Lys48 的 NH3+ 基团被发现具有最高的流动性，其 NMR 分析的六个 NH3+ 基团中具有最低的主链构象参数。我们将实验得到的赖氨酸 NH3+ 基团的主链构象参数与在显式溶剂中进行的 1 μs 分子动力学模拟的结果进行了比较，发现两者之间具有良好的一致性。此外，计算机模拟和 NH3+ 基团键旋转的实验相关时间都表明其氢键具有高度动态性，寿命短于纳秒。本研究证明了将 NMR 实验和模拟相结合用于深入研究这些在功能上最重要的泛素侧链动力学的有效性。
