Collaborative Research: RUI: An In-depth Characterization of Divalent Cadmium Binding to Human Cardiac Troponin C and the Impact on Subsequent Protein Interactions

East Carolina University, Greenville NC

Investigators

Abstract

With the support of the Chemistry of Life Processes Program in the Division of Chemistry, Anne M. Spuches from East Carolina University and Nicholas Grossoehme from Winthrop University will investigate the impact of divalent cadmium, Cd(II), binding to human cardiac troponin C protein (hcTnC) and its impact on subsequent protein interactions. Cadmium toxicity remains an important environmental concern. Chronic cadmium exposure, either from environmental sources or tobacco smoke, can result in numerous health problems including cognitive impairment in children and cardiovascular disease. It is known that Cd(II) can mimic divalent calcium, Ca(II), and can disrupt calcium signaling pathways but little else is known about how this happens at the molecular level. The proposed research aims to investigate Cd(II) binding to human cardiac troponin C, a Ca(II) binding protein responsible for heart muscle contraction, and to explore this toxic metal’s impact on subsequent protein interactions. Furthermore, the proposed project will provide both undergraduate and master’s students the opportunity to engage in independent and meaningful research projects in the field of bioinorganic chemistry. They will be introduced to cutting edge instrumentation and will communicate their findings at national conferences or through publication. These activities will allow students to develop the research and collaborative work skills and the confidence necessary for successful careers in the pharmaceutical or biotechnology industries or in academia. The proposed research uses a combination of calorimetric, spectroscopic, and computational methods to investigate Cd(II) binding to hcTnC and understand how the presence of Cd(II) impacts subsequent protein interactions within the troponin complex. First, Cd(II) binding to the N-terminal domain of human cardiac troponin C and cysteine replacement mutants will be characterized by isothermal titration calorimetry (ITC), NMR, and mass spectrometry methods. Next, site-specific binding of Ca(II) and Cd(II) to EF-hand metal binding knockout mutants in addition to synthesized EF-hand peptides III and IV will be investigated using ITC, fluorescence, and circular dichroism spectroscopy. Finally, hTnC-TnI and TnT protein interactions will be explored in the presence of Cd(II) through the use of surface plasmon resonance (SPR), ITC, and molecular dynamics simulations (MDS). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.