Showcase of Undergraduate Research and Creative Endeavors (SOURCE)

Protein Interactions within the Troponin Complex in Response to Cd2+ Binding

The process of muscle contraction regulation is related and dependent on the Ca²⁺ binding to the protein structure troponin. This is a protein complex composed of three proteins: troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnC is the binding site for Ca²⁺, TnI inhibits the function of muscle contraction and TnT works directly in relation to tropomyosin. The binding of Ca²⁺ to TnC triggers a conformational change that initiates muscle contraction. The long-term goal for this project is to investigate the binding events of a toxic metal, Cadmium and reveal the effects it has on the function of the troponin complex. To achieve this, a short-term goal, which consisted of purifying large amounts of the proteins, TnI was designed. Previous attempts using a common solubility tag known as a maltose binding protein (MBP) allowed the protein to remain soluble; however, when the MBP tag was cleaved off, the isolated TnI protein was insoluble and rapidly precipitated. Our approach consisted of expressing MBP tagged proteins TnT and TnI into Rosetta-2 cells and then purifying the proteins without the treatment of the TEV protease. The cells were lysed, harvested, and centrifuged to isolate our proteins. Affinity chromatography was then used to purify the proteins by introducing a sodium chloride and tris buffer. The identity of the proteins along with some other remnants was confirmed using gel electrophoresis.