Figure 1. Illustration of sample preparation for in-cell NMR experiments. (A) Isotopically (e.g. 13C, 15N, or 19F)-labeled target proteins are overexpressed in host cells (e.g. Escherichia coli) and NMR signals of the target protein (e.g. two-dimenstional 1H-15N heteronuclear single quantum coherence (HSQC) are observed in a NMR spectrum. (B) Isotopically-labeled target protein (the molecule colored by dark gray) and unlabeled partner protein (the molecule with light gray) are concurrently overexpressed in host cells. By comparing the intensity and the chemical shift of NMR signals in spectra with and without over-expression of partner molecules, interaction sites on the target proteins for partners in living cells can be revealed. The numbers of "1" and "2" in the host cell are correspond to the "Signal 1" and "Signal 2" denoted on the schematic illustration of in NMR spectra on right, respectively. In this case, a region around the residue of "Signal 1" should be a binding site as the change in the chemical shift of only "Signal 1" is observed.
Figure 2. Valiation of sample preparation and strategies for in-cell NMR experiments. (A) Isotopically-labeled target proteins are first overexpressed by using an appropriate protein expression system (e.g. Escherichia coli (E. coli)), and purified target proteins are introduced into host cells for in-cell NMR experiments. (Upper left) In the case of oocyte as a host cell, the target proteins are introduced into cells by microinjection. (Upper right) In many cases of mammalian cells, the target proteins are penetrated into cells by using pore-forming toxin (e.g. Streptlysin O (SLO)), (lower right) by performing electroporation, or (lower left) aby fusing a cell-penetrating peptide (CPP) (e.g. Tat peptide) to the target protein. CPP can be eliminated in the host cells when CPP-fused target proteins eneter successfully cells.
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