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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.

Protein Delivery into Plant Cells: Toward In vivo Structural Biology