Utilizing Isothermal Calorimetry to Assess Troponin Complex Stability
Session Title
Science and Technology
College
College of Arts and Sciences
Department
Chemistry, Physics, Geology, & the Environment
Abstract
The regulation of muscle contraction is contingent on Ca 2+ binding to the troponin (Tpn) complex, which is composed of TpnC, TpnI, and TpnT. Each protein serves a distinct role in the regulation strategy: TpnT makes the primary interaction with tropomyosin, TpnI is responsible for the inhibitory interaction that prevents muscle contraction, and TpnC responds to cellular Ca2+ signals, triggering a structural change that abolishes the inhibitory interaction between TpnI and the muscle fiber. The toxic metal, cadmium, has been implicated in impaired cardiac function and it has been proposed by our research team that this may be due to impacting Ca 2+ signals; indeed, previous research shows a strong interaction between cadmium TnC. The goal of this project is to further study the thermodynamic and structural impacts of cadmium binding to the troponin complex. The focus of this project is to design and perform isothermal titration calorimetry (ITC) experiments to establish baseline thermodynamic data for the impact of cadmium binding on the formation of the Tpn complex. Our strategy involved cloning a protease cleavable maltose binding protein affinity tag onto each protein. Once cloned, the construct was over-expressed in E. Coli and purified using modern chromatography techniques. Protease removal of the affinity tag on TpnC presented unexpected complications, so an alternate strategy involving a different affinity tag, hexahistidine, was successfully adopted. Future goals for this project are to design and carry out ITC experiments.
Start Date
15-4-2022 12:00 PM
Utilizing Isothermal Calorimetry to Assess Troponin Complex Stability
The regulation of muscle contraction is contingent on Ca 2+ binding to the troponin (Tpn) complex, which is composed of TpnC, TpnI, and TpnT. Each protein serves a distinct role in the regulation strategy: TpnT makes the primary interaction with tropomyosin, TpnI is responsible for the inhibitory interaction that prevents muscle contraction, and TpnC responds to cellular Ca2+ signals, triggering a structural change that abolishes the inhibitory interaction between TpnI and the muscle fiber. The toxic metal, cadmium, has been implicated in impaired cardiac function and it has been proposed by our research team that this may be due to impacting Ca 2+ signals; indeed, previous research shows a strong interaction between cadmium TnC. The goal of this project is to further study the thermodynamic and structural impacts of cadmium binding to the troponin complex. The focus of this project is to design and perform isothermal titration calorimetry (ITC) experiments to establish baseline thermodynamic data for the impact of cadmium binding on the formation of the Tpn complex. Our strategy involved cloning a protease cleavable maltose binding protein affinity tag onto each protein. Once cloned, the construct was over-expressed in E. Coli and purified using modern chromatography techniques. Protease removal of the affinity tag on TpnC presented unexpected complications, so an alternate strategy involving a different affinity tag, hexahistidine, was successfully adopted. Future goals for this project are to design and carry out ITC experiments.
