NMR insights into the inner workings of living cells.
NMR insights into the inner workings of living cells.
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DOI：
10.1021/ac501467x
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发表时间：
2015-01
影响因子：
7.4
通讯作者：
M. Lerche;P. Jensen;M. Karlsson;S. Meier
中科院分区：
文献类型：
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作者：
M. Lerche;P. Jensen;M. Karlsson;S. Meier
一些最重要的化学问题涉及复杂的分子系统，如活细胞。越来越先进的方法促进了对复杂系统的洞察，或者通过将肉眼看不见的化学过程可视化来实现全新类型的实验。1通过中断、放大和物理分离对细胞组件进行编目的技术已经很成熟。相比之下，对蜂窝组件的功能和相互作用的无破坏性洞察仍然是一个相当大的挑战。然而，在过去的几年里，光谱学已经发展到了复杂的水平，形成了深入了解活细胞内部工作原理的基础。对生命系统的检查通常取决于使用低能量辐射的非破坏性光学或核光谱。核磁共振检测是能量最低的方法，提供了极好的化学分辨率，但目前的仪器灵敏度不高。3通过提高信噪比，大大提高了核磁共振波谱在细胞生物学中的应用范围。这些改进都是通过方法和技术的改进来寻求的，包括增强感兴趣分子中的核磁共振可检测到的核磁性。4这种改进使得对大分子和小分子探针的非侵入性、选择性观察具有更高的时间分辨率和对细胞背景的灵敏度(表1)。以这种方式，核磁共振光谱学不断发展成为一种改进的方法，为细胞生物学提供以前无法获取的信息，包括分子细节和单个原子位置的分辨率。具有不同可视化细胞结构和功能的不同范围的核磁共振平台已经成为现实，包括(1)使用细胞作为试管的高分辨率细胞内核磁共振，以确定细胞环境中的大分子结构、翻滚、动力学和相互作用；(2)全细胞制剂或结构的固态核磁共振谱以及在细胞表面或天然膜中的相互作用；(3)通过与内源性小分子探针的细胞反应和高分辨率核磁共振检测，测量细胞生理参数(如pH、[Ca2+]、氧化还原状态)和动态(时间分辨)的代谢产物通量。利用这些平台，已经开发了各种应用，用于探测大分子结构、动力学以及与活细胞的液态核磁共振波谱和与细胞制剂的固态核磁共振波谱的相互作用。表2给出了这些应用的概述。最近汇编了关于大分子16−18的细胞内和细胞内核磁共振的全面综述。因此，在这里，我们更具体地关注当前对活细胞内部工作原理的基本洞察的希望和挑战，使用核磁共振可检测的探针，这些探针是由最大化被检测分子信号的“超极化方法”产生的。14，15超极化是一种物理自旋有序方法的统称，这种方法直接或通过将核磁矩耦合到其他具有高极化的自旋，暂时将核磁共振可检测到的核磁性提高几个数量级(图1)。已经描述了各种超极化方法。37−40，而超极化方法的理论基础不是
Some of the most important chemical problems involve complex molecular systems such as living cells. Increasingly advanced methodologies facilitate insights into complex systems or enable entirely new types of experiments by visualizing chemical processes that are invisible to the naked eye. 1 Technologies for the cataloguing of cellular components by disruption, amplification, and physical separation are well established. In contrast, nondisruptive insights into the functions and interactions of cellular components remain a considerable challenge. 2 Within the past few years, however, spectroscopy has evolved to levels of sophistication that form the basis for gaining insights into the inner workings of the living cell. The scrutiny of living systems usually hinges upon nondestructive optical or nuclear spectroscopy using lowenergy radiation. Nuclear magnetic resonance (NMR) detection is the lowest-energy method and provides excellent chemical resolution, but current instrumentation is not highly sensitive. 3The scope of NMR spectroscopy for cell biology greatly improves by increasing the signal-to-noise ratio. These improvements are sought both through methodological and technological improvements, including the enhancement of NMR detectable nuclear magnetism in the molecules of interest. 4 Such improvements have permitted the noninvasive, selective observation of macromolecules and small molecular probes with enhanced time resolution and sensitivity against cellular backgrounds (Table 1). In this manner, NMR spectroscopy keeps developing into an improved method yielding previously inaccessible information for cell biology with molecular detail and resolution of individual atomic sites. NMR platforms with different scopes for visualizing cellular structures and functions have materialized, including (1) highresolution in-cell NMR of macromolecules using cells as test tubes for determining macromolecular structure, tumbling, dynamics, and interactions in cellular environments;(2) solidstate NMR spectra of whole cell preparations or structures and interactions at the cell surface or in native membranes;(3) the measurement of cell physiological parameters (eg, pH,[Ca2+], redox state) and dynamic (time-resolved) tracking of metabolite fluxes through cellular reactions with endogenous small molecule probes and high-resolution NMR detection. Using these platforms, a variety of applications have been developed for the probing of macromolecular structure, dynamics, and interactions with liquid state NMR spectroscopy of living cells and with solid-state NMR spectroscopy of cell preparations. An overview of these applications is given in Table 2. Comprehensive reviews covering in-cell and on-cell NMR of macromolecules 16− 18 have recently been assembled. Here, we therefore focus more specifically on the current promises and challenges for fundamental insights into the inner workings of living cells, using NMR detectable probes generated by “hyperpolarization methods” that maximize the signal of the detected molecules. 14, 15 Hyperpolarization is a collective term for a physical spin ordering approach that temporarily enhances NMR-detectable nuclear magnetism by several orders of magnitude, either directly or through the coupling of nuclear magnetic moments to other spins with high polarization (Figure 1). Various hyperpolarization methodologies have been described. 37− 40 While the theoretical basis for hyperpolarization methods is not