DOI: 10.1101/455063Oct 30, 2018Paper

Fusion of DARPin to aldolase enables visualization of small protein by cryoEM

BioRxiv : the Preprint Server for Biology
Qing YaoGrant J Jensen

Abstract

In recent years, solving protein structures by single particle cryogenic electron microscopy (cryoEM) has become a crucial tool in structural biology. While exciting progress is being made towards the visualization of smaller and smaller macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of single particle cryoEM. To overcome this problem, we implemented a platform strategy in which a small protei target was rigidly attached to a large, symmetric base via a selectable adapter. Seven designs were tested. In the best construct, a designed ankyrin repeat protein (DARPin) was rigidly fused to tetrameric rabbit muscl aldolase through a helical linker. The DARPin retained its ability to bind its target, the 27 kDa green fluorescent protein (GFP). We solve the structure of this complex to 3.0 Å resolution overall, with 5 to 8 Å resolution in the GFP region. As flexibility in the DARPin limited the overall resolution of the target, we described strategies to rigidify this element.

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