Protein Delivery into Plant Cells: Toward In vivo Structural Biology.
Protein Delivery into Plant Cells: Toward In vivo Structural Biology.
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DOI：
10.3389/fpls.2017.00519
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发表时间：
2017
影响因子：
5.6
通讯作者：
Tompa P
中科院分区：
文献类型：
--
作者：
Cedeño C;Pauwels K;Tompa P
了解活植物细胞内蛋白质的生物学相关结构和功能行为只有通过结构生物学和细胞生物学的结合才有可能。最先进的结构生物学技术通常应用于从其原生环境中分离出来的分子。虽然在处理分离纯化的蛋白质时，大多数实验条件可以很容易地控制，但这种体外工作的一个严重缺点是我们无法模拟蛋白质存在和功能的极其复杂的细胞内环境。因此，在蛋白质的自然栖息地，即活细胞内研究蛋白质是非常可取的。这是细胞内核磁共振的主要目标，其目的是在生理条件下将标记蛋白传递到细胞后，在真实的体内条件下接近结构-功能关系。通过多学科方法，包括重组蛋白生产，共聚焦荧光显微镜，核磁共振（NMR）光谱和不同的细胞内蛋白质递送策略，我们探索在活植物细胞中开展细胞内核磁共振研究的可能性。虽然我们提供了一个全面的框架来建立细胞内核磁共振，但我们确定了同位素标记蛋白在细胞内的有效引入是主要的瓶颈。基于典型的内在紊乱蛋白（IDPs）对脱水蛋白10和14的早期反应实验，我们还建立了ERD14在非生物胁迫下的亚细胞定位。
Understanding the biologically relevant structural and functional behavior of proteins inside living plant cells is only possible through the combination of structural biology and cell biology. The state-of-the-art structural biology techniques are typically applied to molecules that are isolated from their native context. Although most experimental conditions can be easily controlled while dealing with an isolated, purified protein, a serious shortcoming of such in vitro work is that we cannot mimic the extremely complex intracellular environment in which the protein exists and functions. Therefore, it is highly desirable to investigate proteins in their natural habitat, i.e., within live cells. This is the major ambition of in-cell NMR, which aims to approach structure-function relationship under true in vivo conditions following delivery of labeled proteins into cells under physiological conditions. With a multidisciplinary approach that includes recombinant protein production, confocal fluorescence microscopy, nuclear magnetic resonance (NMR) spectroscopy and different intracellular protein delivery strategies, we explore the possibility to develop in-cell NMR studies in living plant cells. While we provide a comprehensive framework to set-up in-cell NMR, we identified the efficient intracellular introduction of isotope-labeled proteins as the major bottleneck. Based on experiments with the paradigmatic intrinsically disordered proteins (IDPs) Early Response to Dehydration protein 10 and 14, we also established the subcellular localization of ERD14 under abiotic stress.