Using Genetically Encodable Self-Assembling GdIIISpin 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 GdIIImetal-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 GdIIIlabels 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.