Conserved linker length in double dsRBD proteins from plants restricts interdomain motion

Double stranded RNA binding domains (dsRBDs) are ubiquitous in all kingdoms of life. They can participate both in RNA and protein recognition and are usually present in multiple copies in multidomain proteins. We analyzed the linkers between dsRBDs in different proteins and found that sequences corresponding to plant proteins have a highly conserved linker length. In order to assess the importance of linker length in the conformational freedom of double dsRBD plant proteins, we introduced lanthanide binding tags (LBTs) in different positions of the dsRBD containing protein HYL1 from Arabidopsis thaliana. These constructs were used to obtain conformational restraints from Double electron–electron resonance (DEER) measurements on doubly labeled proteins and from paramagnetic relaxation enhancement (PRE) in single labeled samples. Fitting the experimental datasets to a computational model of the ensemble created by allowing freedom to the linker region we found that the domains tend to explore a particular region of the allowed conformational space. The high conservation in linker length suggests that this restricted conformational sampling is functional, possibly hindering HYL1-dsRBD2 from contacting the substrate dsRNA and allowing it to participate in protein-protein interactions

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

International audienceDouble 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

The Use of Mn(II) Bound to His-tags as Genetically Encodable Spin-Label for Nanometric Distance Determination in Proteins

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