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Angew Chem Int Ed Engl. 2016 Sep 5;55(37):11041-3. doi: 10.1002/anie.201603653. Epub 2016 Aug 5.

Using Genetically Encodable Self-Assembling Gd(III) Spin Labels To Make In-Cell Nanometric Distance Measurements.

Author information

1
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.
2
Institut de Biologie Intégrative de la Cellule (I2BC), IBITECS, CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, F-91198, Gif-sur-Yvette, France.
3
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.

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.

KEYWORDS:

EPR spectroscopy; gadolinium; in cell spectroscopy; protein structures; spin labels

PMID:
27496179
DOI:
10.1002/anie.201603653
[Indexed for MEDLINE]

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