2015 REU Poster: Cloning, Expression and Purification of HisSumoMet18TevStrep, and Chemical Crosslinking of Proteins in the targeting Complex of Cytosolic Iron Sulfur Cluster Pathway
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Iron sulfurs play an important role in many biological functions such as: DNA replication, and repair, amino acid metabolism, and respiration. In yeast, the 4Fe-4S cluster needed by proteins located in the nucleus and in the cytosol are assembled and inserted by the Cytosolic Iron Sulfur Cluster Assembly (CIA) pathway. These clusters are assembled by the scaffold and sent to inactive Apo proteins (without clusters) by the targeting complex. In the cytosol, there is a three protein complex called the CIA targeting complex that identifies apo-targets in order to assemble them to their cofactor. This targeting complex comprised of the protein Cia 1, Cia 2, and met18 which are yeast proteins. However, the mechanism by which this occurs has not been established. One thing that has limited our ability to understand apo-enzyme recognitions has been problems that has to do with purification of the individual components of the CIA targeting complex. Previous work has shown that purification of Met18 is challenging due to purification of many undesired truncated products. To optimize purification of the full length protein, I designed a construct with a C-terminal Strep tag. Another challenge faced is the weak and dynamic interaction that exists between the CIA targeting components and apo-proteins. To stabilize these interactions so they can be characterized, I developed a method to covalently crosslink Cia1 to Cia 2 The pure full length Met18 was purified using the new construct that was purified using the new construct, and using a streptavidin affinity resin. Also, the mixture of both Cia 1 and Cia 2 formed what looks like a dimer and a monomer. Cia 2 alone also crosslinked, while Cia 1 alone did not have any crosslinked product which was what we expected because of the prior knowledge we have about how they exist. Now with sufficient amounts of each component of the targeting complex at our disposal, the interactions between these proteins can now be defined, and to help with biochemical characterization, crosslinking Cia1 and Cia2 using DSS crosslinker shows that the dynamic interactions that exists between the trafficking complex components can be stabilized using DSS crosslinker.
Poster presentation at REU Summer's End Research Symposium, 2015, by REU participant Zanub Hassan, Harry S. Truman Community College - Deborah Perlstein group, Amanda Vo lab mentor
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