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

合作研究：RUI：二价镉与人心肌肌钙蛋白 C 结合的深入表征以及对后续蛋白质相互作用的影响

• 批准号: 2203467
• 负责人: Nicholas Grossoehme
• 金额: $ 11.82万
• 依托单位: Winthrop University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2203467&HistoricalAwards=false
• 关键词: Collaborative; Research; RUI; depth; Characterization

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.

在化学系生命过程化学项目的支持下，东卡罗莱纳大学的Anne M. Spuches和温斯洛普大学的Nicholas Grossoehme将研究二价镉Cd（II）与人类心脏肌钙蛋白C （hcTnC）结合的影响及其对随后蛋白质相互作用的影响。镉毒性仍然是一个重要的环境问题。慢性镉暴露，无论是来自环境来源还是来自烟草烟雾，都可导致许多健康问题，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)，并且可以破坏钙信号通路，但对于这在分子水平上是如何发生的知之甚少。拟议的研究旨在研究Cd（II）与人心脏肌钙蛋白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随后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。他们将被介绍最前沿的仪器，并将在国家会议上或通过出版物交流他们的发现。这些活动将使学生能够发展研究和协作工作技能，以及在制药或生物技术行业或学术界取得成功所必需的信心。这项拟议的研究结合了量热法、光谱法和计算方法来研究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物内随后的蛋白质相互作用。首先，Cd（II）结合到人类心脏肌钙蛋白C和半胱氨酸替代突变体的n端结构域将通过等温滴定量热法（ITC）、核磁共振和质谱方法进行表征。接下来，将使用ITC、荧光和圆二色光谱研究Ca（II）和Cd（II）与EF-hand金属结合敲除突变体以及合成的EF-hand肽III和IV的位点特异性结合。最后，hTnC-TnI和TnT蛋白在Cd（II）存在下的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）来探索。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。