Fig. 4.

Overview of intracellular protein delivery methods for in cell structural analysis (mainly using NMR). a The IDP of interest can be overexpressed with recombinant DNA technology in prokaryotic or eukaryotic cells by growing the cells in unlabeled medium, followed by induction of expression in isotopically labelled medium. The cell suspension can be readily used for direct in cell NMR data collection. b Alternatively, the protein of interest is overexpressed using recombinant production systems, purified to homogeneity and introduced into (eukaryotic) cells via either microinjection [e.g. a Xenopus laevis oocyte is kept in place with a holding pipet (represented in blue) while the purified IDP is injected intracellularly]. c Electroporation causes transient permeability of the cell membrane to allow the protein of interest to enter. d Cell penetrating peptides (CPP, represented in red) that are covalently linked to the proteins allow entry of the protein inside the cells, and CPPs can be removed subsequently. e IDPs can be delivered through cell-permeabilizing toxins that form transient pores, which enable the protein to diffuse into the cell according to the concentration gradient. As an IDP example in a and b, the ensemble of monomeric α-synuclein is represented by ten different conformational states that were generated using molecular dynamics simulations restrained with inter-atomic distances derived from paramagnetic relaxation enhancement NMR (PED entry 9AAC)