Visualizing Small Proteins with the cryoEM Platform and The Structure of the Vibrio cholerae Type IV Competence Pilus Secretin PilQ

Citation

Weaver, Sara Jean (2020) Visualizing Small Proteins with the cryoEM Platform and The Structure of the Vibrio cholerae Type IV Competence Pilus Secretin PilQ. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/9B9V-PK08. https://resolver.caltech.edu/CaltechTHESIS:12092019-141850032

Abstract

Solving protein structures by single-particle cryoelectron microscopy (cryo-EM) has become a crucial tool in structural biology. While exciting progress is being made toward the visualization of small macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of cryo-EM. To overcome this problem, we implemented a platform strategy in which a small protein target was rigidly attached to a large, symmetric base via a selectable adapter. Of our seven designs, the best construct used a designed ankyrin repeat protein (DARPin) rigidly fused to tetrameric rabbit muscle aldolase through a helical linker. The DARPin retained its ability to bind its target: GFP. We solved the structure of this complex to 3.0 Å resolution overall, with 5-8 Å resolution in the GFP region. As flexibility in the DARPin position limited the overall resolution of the target, we describe strategies to rigidify this element.

Natural competence is the process by which bacteria take up genetic material from their environment and integrate it into their genome using homologous recombination. In Vibrio cholerae, the Type IV competence pilus is thought to mediate DNA uptake by binding DNA and retracting back toward the cell. How the DNA enters the periplasm is unclear. One hypothesis suggests that the DNA-bound Type IV competence pilus retracts completely so that the DNA would pass through the outer membrane secretin pore (PilQ). PilQ is a 870 kDa outer membrane pore with C14 symmetry. Here, we purify the V. cholerae PilQ secretin from V. cholerae cells in amphipols for single particle cryogenic electron microscopy (cryoEM). We solve the structure to 3.0 Å and provide insight on the channel DNA may traverse through during uptake.

Thesis Files