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，东卡罗来纳大学的Anne M.Spucches和温斯罗普大学的Nicholas Grossoehme将研究二价镉Cd(II)与人心肌肌钙蛋白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。慢性接触镉，无论是来自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。众所周知，Cd(II)可以模拟二价钙，Ca(II)，并可以扰乱钙信号通路，但对分子水平上如何发生这种情况知之甚少。这项拟议的研究旨在研究Cd(II)与人心肌肌钙蛋白C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，拟议的项目将为本科生和硕士学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。他们将被介绍给尖端仪器，并将在国家会议上或通过出版物传达他们的研究结果。这些活动将使学生培养在制药或生物技术行业或在学术界成功职业所必需的研究和协作技能以及信心。拟议的研究结合量热、光谱和计算方法来研究Cd(II)与hcTnC的结合，并了解Cd(II)的存在如何影响肌钙蛋白复合体中后续的蛋白质相互作用。首先，结合到人心肌肌钙蛋白C和半胱氨酸替换突变体N-末端的Cd(II)将用等温滴定热法(ITC)、核磁共振和质谱学方法进行表征。接下来，除了合成EF-手肽III和IV外，还将利用ITC、荧光和圆二色光谱研究Ca(II)和Cd(II)与EF-手性金属结合敲除突变体的特异性结合。最后，将通过使用表面等离子体共振(SPR)、ITC和分子动力学模拟(MDS)来探索在Cd(II)存在的情况下hTnC-TnI和TNT蛋白质的相互作用。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.