title: In-cell NMR in mammalian cells: part 1.
abstract: Many mammalian IDPs exert important biological functions in key cellular processes and often in highly specialized subsets of cells. For these reasons, tools to characterize the structural and functional characteristics of IDPs inside mammalian cells are of particular interest. Moving from bacterial and amphibian in-cell NMR experiments to mammalian systems offers the unique opportunity to advance our knowledge about general IDP properties in native cellular environments. This is never more relevant than for IDPs that exhibit pathological structural rearrangements under certain cellular conditions, as is the case for human α-synuclein in dopaminergic neurons of the substantia nigra in the course of Parkinson's disease, for example. To efficiently deliver isotope-labeled IDPs into mammalian cells is one of the first challenges when preparing a mammalian in-cell NMR sample. The method presented here provides a detailed protocol for the transduction of isotope-labeled α-synuclein, as a model IDP, into cultured human HeLa cells. Cellular IDP delivery is afforded by action of a cell-penetrating peptide (CPP) tag. In the protocol outlined below, the CPP tag is "linked" to the IDP cargo moiety via an oxidative, disulfide-coupling reaction.
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: Alexander
authors: A
authors: Dose
authors: Dirk
authors: D
authors: Schwarzer
authors: Philipp
authors: P
authors: Selenko
mesh_terms: D057846:Cell-Penetrating Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D058451:Oxidative Coupling; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D051844:alpha-Synuclein / Q000737:chemistry*
chemical_list: D057846:Cell-Penetrating Peptides; D011994:Recombinant Proteins; D051844:alpha-Synuclein