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

在化学系生命过程化学项目的支持下，Anne M.来自东卡罗莱纳大学的Spuches和来自温斯洛普大学的Nicholas Grossoehme将研究二价镉（Cd（II））与人类心肌肌钙蛋白C蛋白（hcTnC）结合的影响及其对后续蛋白质相互作用的影响。镉的毒性仍然是一个重要的环境问题。长期接触镉，无论是环境来源还是烟草烟雾，都可能导致许多健康问题，包括儿童认知障碍和心血管疾病。已知Cd（II）可以模拟二价钙，Ca（II），并可以破坏钙信号通路，但很少有人知道这是如何在分子水平上发生的。拟议的研究旨在研究Cd（II）与人体心肌肌钙蛋白C（一种负责心肌收缩的Ca（II）结合蛋白）的结合，并探索这种有毒金属对后续蛋白质相互作用的影响。此外，该项目将为本科生和硕士生提供在生物无机化学领域从事独立和有意义的研究项目的机会。将向他们介绍最先进的仪器，并在国家会议上或通过出版物传达他们的研究结果。这些活动将使学生发展必要的研究和协作工作技能和信心，在制药或生物技术行业或在academy.The拟议的研究使用量热，光谱和计算方法相结合，以调查镉（II）结合hcTnC和了解镉（II）的存在如何影响随后的蛋白质相互作用内的肌钙蛋白复合物。首先，镉（II）结合的N-末端结构域的人心肌肌钙蛋白C和半胱氨酸置换突变体将其特征在于等温滴定量热法（ITC），NMR和质谱法。接下来，除了合成EF-手肽III和IV外，还将使用ITC、荧光和圆二色光谱研究Ca（II）和Cd（II）与EF-手金属结合敲除突变体的位点特异性结合。最后，hTnC-TnI和TnT蛋白相互作用将通过使用表面等离子体共振（SPR），ITC和分子动力学模拟（MDS）在Cd（II）的存在下进行探索。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.