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

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, invitro 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 super(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 super(III)-supplemented medium. DEER measurements directly on these cells produced readily detectable time traces from which the distance between the Gd super(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.Original Abstract: Reservierte Plaetze: Ein Dreihelixbuendel, das mit zwei flankierenden Lanthanoid-Bindestellen markiert ist, wurde in E.coli exprimiert. Dem Medium zugesetztes Gd super(III) kann in die Zellen eindringen und durch Besetzen der Bindestellen das biosynthetische spinmarkierte Protein vervollstaendigen. DEER-Messungen an intakten Zellen belegen die Eignung des Ansatzes fuer die Untersuchung von Proteinstrukturen unter nativen Bedingungen.