title: Using Genetically Encodable Self-Assembling Gd(III) Spin Labels To Make In-Cell Nanometric Distance Measurements.
abstract: Double electron-electron resonance (DEER) can be used to study the structure of a protein in its native cellular environment. Until now, this has required isolation, in vitro labeling, and reintroduction of the protein back into the cells. We describe a completely biosynthetic approach that avoids these steps. It exploits genetically encodable lanthanide-binding tags (LBT) to form self-assembling Gd(III) metal-based spin labels and enables direct in-cell measurements. This approach is demonstrated using a pair of LBTs encoded one at each end of a 3-helix bundle expressed in E. coli grown on Gd(III) -supplemented medium. DEER measurements directly on these cells produced readily detectable time traces from which the distance between the Gd(III) labels could be determined. This work is the first to use biosynthetically produced self-assembling metal-containing spin labels for non-disruptive in-cell structural measurements.
authors: Instituto de Biología Molecular y Celular de Rosario; Área Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Ocampo y Esmeralda, Rosario, 2000, Argentina.
authors: Florencia C
authors: FC
authors: Mascali
authors: Institut de Biologie Intégrative de la Cellule (I2BC), IBITECS, CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, F-91198, Gif-sur-Yvette, France.
authors: H Y Vincent
authors: HY
authors: Ching
authors: Instituto de Biología Molecular y Celular de Rosario; Área Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Ocampo y Esmeralda, Rosario, 2000, Argentina.
authors: Rodolfo M
authors: RM
authors: Rasia
authors: Institut de Biologie Intégrative de la Cellule (I2BC), IBITECS, CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, F-91198, Gif-sur-Yvette, France.
authors: Sun
authors: S
authors: Un
authors: Institut de Biologie Intégrative de la Cellule (I2BC), IBITECS, CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, F-91198, Gif-sur-Yvette, France. leandro.tabares@cea.fr.
authors: Leandro C
authors: LC
authors: Tabares
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000166:cytology* / Q000254:growth & development; D005682:Gadolinium / Q000737:chemistry*; D010316:Particle Size; D013113:Spin Labels
chemical_list: D013113:Spin Labels; D005682:Gadolinium
keywords: EPR spectroscopy; gadolinium; in cell spectroscopy; protein structures; spin labels