Investigation of Protein-Protein Interactions of the Troponin Complex in Response to Ca2+ and Cd2+ binding
Session Title
Additional Projects
College
College of Arts and Sciences
Department
Chemistry, Physics, Geology, & the Environment
Abstract
Troponin is a protein complex present in the thin filaments of all muscles. Its primary role is to facilitate muscle contraction in response to Ca2+ signals that originate from nerve impulses. It is composed of three distinct proteins: troponin I (TnI), which inhibits actin-myosin interaction; troponin T (TnT), which provides the primary interaction with tropomyosin; and troponin C (TnC), which binds Ca2+ ions, which is the crucial step of the contracting mechanism. Recent evidence suggests that the toxic metal cadmium, Cd2+, can compete calcium binding to TnC. The goal of this project is to investigate the impact of Cd2+-binding to TnC on the formation and stability of the troponin complex. Surface Plasmon Resonance (SPR) will be used to study the protein-protein interactions between troponin complex proteins and characterize how these interactions respond in the presence of the two metals. Previously, our research team has designed purification strategies for each of the troponin proteins; however, solubility issues were encountered during the final step of TnI and TnT purification. Briefly, the purification system was designed to incorporate a protease cleavable MBP fusion protein to the N-terminus of TnI or TnT. Upon proteolysis, it was found that these proteins are not soluble in the absence of the MBP tag. Presently, the approach has been modified to incorporate a second tag, monomeric streptavidin (mSA), between the MBP and TnI/T. We hypothesize that this tag will increase the solubility of the proteins while providing an ideal anchor for SPR chip attachment.
Start Date
15-4-2022 12:00 PM
Investigation of Protein-Protein Interactions of the Troponin Complex in Response to Ca2+ and Cd2+ binding
Troponin is a protein complex present in the thin filaments of all muscles. Its primary role is to facilitate muscle contraction in response to Ca2+ signals that originate from nerve impulses. It is composed of three distinct proteins: troponin I (TnI), which inhibits actin-myosin interaction; troponin T (TnT), which provides the primary interaction with tropomyosin; and troponin C (TnC), which binds Ca2+ ions, which is the crucial step of the contracting mechanism. Recent evidence suggests that the toxic metal cadmium, Cd2+, can compete calcium binding to TnC. The goal of this project is to investigate the impact of Cd2+-binding to TnC on the formation and stability of the troponin complex. Surface Plasmon Resonance (SPR) will be used to study the protein-protein interactions between troponin complex proteins and characterize how these interactions respond in the presence of the two metals. Previously, our research team has designed purification strategies for each of the troponin proteins; however, solubility issues were encountered during the final step of TnI and TnT purification. Briefly, the purification system was designed to incorporate a protease cleavable MBP fusion protein to the N-terminus of TnI or TnT. Upon proteolysis, it was found that these proteins are not soluble in the absence of the MBP tag. Presently, the approach has been modified to incorporate a second tag, monomeric streptavidin (mSA), between the MBP and TnI/T. We hypothesize that this tag will increase the solubility of the proteins while providing an ideal anchor for SPR chip attachment.
