Figure 1.

Schematic work flow representation to develop an in-cell NMR strategy to study proteins within plant cells. The first considerations and planning of the in-cell NMR experiments should be based on readily available NMR data. It is desirable that the protein of interest has been characterized in vitro by NMR and that a chemical shift assignment is available that ultimately can be used to analyze the in-cell NMR dataset. Since NMR is a high resolution technique, it can address specific questions with regard to structural and dynamical aspects at the residue-level of the protein intracellular behavior. The major experimental obstacle will be the intracellular delivery of isotope-labeled protein. The success of the delivery strategy should entice monitoring the subcellular localization, intracellular protein quantification and the protein integrity. These are also 3 important parameters to check upon completing the actual in-cell NMR data acquisition. Likewise, it is imperative to verify the cell viability and to demonstrate that the protein has not leaked into the extracellular medium so that the NMR signals truly originate from the intracellular protein (Waudby et al., 2012). Another aspect to consider is the occurrence of stress to the cells during the data acquisition, which might be unveiled by an in depth proteomics analysis. To maintain the plant cell suspension in a living state, the experiment can be performed in a bioreactor that was developed for in-cell protein NMR. The performance and capabilities of such a bioreactor has been demonstrated by the overexpression of the intrinsically disordered human protein α-synuclein in Escherichia coli (Sharaf et al., 2010). Ultimately the in vitro NMR assignment should be transferred to the in-cell NMR datasets and employed to answer the relevant questions.