title: In-cell NMR in mammalian cells: part 2.
abstract: Delivery of isotope-labeled IDPs into mammalian cells for the purpose of generating suitable in-cell NMR samples can also be facilitated by action of pore-forming bacterial toxins. In the course of this procedure, mammalian cell membranes are permeated for short periods of time in order to enable the influx of exogenous proteins via a concentration gradient between the outside and the inside of the targeted "host" cells. In contrast to CPP-mediated IDP uptake, toxins offer the advantage that cellular protein transduction does not rely on active biological processes like endocytosis, but on simple passive diffusion. Therefore, proteins that are to be delivered into mammalian cells are not required to contain additional "targeting" sequences, and can be employed in their native contexts. The protocol outlined here employs isotope-labeled human α-synuclein, adherent human HeLa cells, and the Streptococcus pyogenes endotoxin Streptolysin O (SLO).
authors: Department of NMR-assisted Structural Biology, Leibniz Institute of Molecular Pharmacology (FMP Berlin), Berlin, Germany.
authors: Beata
authors: B
authors: Bekei
authors: Honor May
authors: HM
authors: Rose
authors: Michaela
authors: M
authors: Herzig
authors: Philipp
authors: P
authors: Selenko
mesh_terms: D001426:Bacterial Proteins / Q000494:pharmacology; D002021:Buffers; D002463:Cell Membrane Permeability / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013301:Streptolysins / Q000494:pharmacology; D051844:alpha-Synuclein / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D002021:Buffers; D011994:Recombinant Proteins; D013301:Streptolysins; D051844:alpha-Synuclein; C025964:streptolysin O