Production, Purification, and X-Ray Crystallography of the POTRA Domains of PsToc75

Plastids arose via endosymbiosis when a cyanobacterium was engulfed by a primitive eukaryote. The cyanobacterium was enslaved by the eukaryote, eventually giving rise to a new organelle, termed the plastid. The transition from a free-living cyanobacterium to a cell-dependent organelle demonstrates how the chloroplast ancestor underwent many changes in its physiology and biological processes. The majority of the DNA belonging to the cyanobacterium was scavenged by the nucleus of the host cell. This led to higher fidelity of genetic duplication, due to the proofreading abilities of the DNA polymerase of the host cell. This left the cell with the problem of how to get the now cytosolically transcribed proteins back into the proto-plastid. Eukaryotic photosynthetic cells use the Translocon(s) of the Outer/Inner envelope of the Chloroplast (TOC/TIC) to import proteins necessary for the survival of the plastid. Although much has been uncovered about the machinery necessary for protein import, the mechanism(s) used to accomplish this import remains unclear. Each translocon comprises a pore, through which precursor proteins are translocated along with accessory proteins that assist in translocation. Toc75 forms the pore in the outer envelope of chloroplasts. Toc75 is a member of the Outer Membrane Protein of 85-kilodalton/Two Partner Secretion (OMP85/TPS) superfamily, which all share an architecture composed of a central membrane channel and cytosolic POTRA (Polypeptide Transport Associated) domains. Structures of POTRA domains from other members of OMP85/TPS have been solved, but the structure of the POTRA domains of Toc75 have not. These POTRA domains are of particular interest because Toc75 interacts with thousands of proteins, far more than other members of the OMP85/TPS. In our project, we have recombinantly expressed the N-terminal POTRA domains of Toc75 insolubly. The POTRA domains were purified via IMAC and have been refolded. Their purity has been accessed by SDS-PAGE. Their secondary structure has been confirmed with circular dichroism, and the peptides are ready to be shipped to our collaborator Dr. Susan Buchanan. (Supported by National Science Foundation.