Dynamics of Lysine Side-Chain Amino Groups in a Protein Studied by Heteronuclear 1H-15N NMR Spectroscopy
Dynamics of Lysine Side-Chain Amino Groups in a Protein Studied by Heteronuclear 1H-15N NMR Spectroscopy
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DOI：
10.1021/ja107847d
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发表时间：
2011-02-02
影响因子：
15
通讯作者：
Iwahara, Junji
中科院分区：
文献类型：
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作者：
Esadze, Alexandre;Li, Da-Wei;Iwahara, Junji
尽管赖氨酸侧链氨基酸在大分子相互作用和功能中具有重要意义，但蛋白质中赖氨酸侧链氨基酸的动力学尚不清楚。在这项研究中，我们开发了一种通过核磁共振光谱和计算来研究链上NH3+基团动力学的方法。通过优化(NH3+)-N-15基团的H-1-N-15异核相关实验，分析了在2℃和pH 5条件下，人泛素中赖氨酸NH3+基团的动态行为。我们修改了先前建立的CH3基团的理论框架，并使用它来分析NH3+基团的N-15弛豫数据。对于泛素中7个赖氨酸NH3+基团中的6个，我们确定了无模型顺序参数，键旋转的相关时间，以及发生在皮到纳秒时间尺度上的对称轴重定向。从赖氨酸NH3+基团的CPMG弛豫色散实验中，也检测到Lys27在毫秒时间尺度上发生了较慢的动力学。Lys48的NH3+基团在蛋白酶体降解的泛素化过程中起着关键的连锁位点作用，在核磁共振分析的6个NH3+基团中，NH3+基团具有较高的流动性，其序参数最低。我们将赖氨酸NH3+的实验序参量与1 μ s分子动力学模拟结果进行了比较，发现两者吻合较好。此外，NH3+基团键旋转的计算机模拟和实验相关时间表明，它们的氢键具有亚纳秒寿命的高动态。这项研究证明了结合核磁共振实验和模拟的效用，深入表征这些功能上最重要的泛素侧链的动力学。
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 lisine NH3+ groups by NMR spectroscopy and computation. By using H-1-N-15 heteronuclear correlation experiments optimized for (NH3+)-N-15 moieties, we have analyzed the dynamic behavior of individual lysine NH3+ groups in human ubiquitin at 2 degrees C and pH 5. We modified the theoretical framework developed previously for CH3 groups and used it to analyze N-15 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 mu s 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.