Using Genetically Encodable Self-Assembling GdIII Spin Labels To Make In-Cell Nanometric Distance Measurements
Florencia C. Mascali
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
Search for more papers by this authorDr. H. Y. Vincent Ching
Institut de Biologie Intégrative de la Cellule (I2BC), IBITECS, CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, F-91198 Gif-sur-Yvette, France
Search for more papers by this authorDr. Rodolfo M. Rasia
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
Search for more papers by this authorDr. Sun Un
Institut de Biologie Intégrative de la Cellule (I2BC), IBITECS, CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, F-91198 Gif-sur-Yvette, France
Search for more papers by this authorCorresponding Author
Dr. Leandro C. Tabares
Institut de Biologie Intégrative de la Cellule (I2BC), IBITECS, CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, F-91198 Gif-sur-Yvette, France
Search for more papers by this authorFlorencia C. Mascali
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
Search for more papers by this authorDr. H. Y. Vincent Ching
Institut de Biologie Intégrative de la Cellule (I2BC), IBITECS, CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, F-91198 Gif-sur-Yvette, France
Search for more papers by this authorDr. Rodolfo M. Rasia
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
Search for more papers by this authorDr. Sun Un
Institut de Biologie Intégrative de la Cellule (I2BC), IBITECS, CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, F-91198 Gif-sur-Yvette, France
Search for more papers by this authorCorresponding Author
Dr. Leandro C. Tabares
Institut de Biologie Intégrative de la Cellule (I2BC), IBITECS, CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, F-91198 Gif-sur-Yvette, France
Search for more papers by this authorAbstract
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 GdIII 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 GdIII-supplemented medium. DEER measurements directly on these cells produced readily detectable time traces from which the distance between the GdIII 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.
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