The Mechanism of Polyvalent Ion Competition with Membranes and Membrane-Associated Proteins

有了这个奖项，生命过程化学计划正在资助马里兰州大学的Jeffery Klauda和Sergei Sukharev博士研究多价离子与脂质和膜相关蛋白的结合竞争。溶液中的钙离子作为信号传导剂和辅助因子发挥着多种关键作用，介导肌肉收缩、神经信号传递、血液凝固，以及重要的死细胞去除。带正电荷的钙离子通过特殊的生物过程帮助识别受损和垂死的细胞，这取决于钙与细胞死亡期间暴露的带负电荷的脂质分子的结合。特定的蛋白质识别这些钙包被的垂死（凋亡）细胞，并且是去除这些细胞的关键。然而，一些有毒的金属离子，如铍，可以竞争，取代和抑制钙介导的自然功能。Jeffery Klauda和Sergei Sukharev博士使用实验和计算相结合的方法来了解钙和铍与模型化合物，脂质和蛋白质的结合和竞争的基本方面。该项目深入了解了铍如何干扰多种钙依赖性过程并特别破坏体内凋亡细胞识别和相关清洁级联反应的机制。该项目为本科生和研究生研究人员提供培训，并通过在科学会议上的介绍和出版物传播研究成果。该项目通过在少数群体服务机构为高中生提供暑期研究实习机会，开展教育推广活动。一系列的磁铁实习将允许学生在Klauda和Sukharev博士的实验室积极进行监督研究。此外，课程讲座和项目研究的实际例子被整合到本科生和研究生的工程和科学课程中，以增加参与度和曝光度。探索离子与细胞膜中脂质结合的结构和热力学一直具有挑战性。该项目旨在使用等温滴定量热法（ITC）来获得钙（II）和铍（II）的结合自由能，以模拟脂质和肽羰基/羧酸酯的化合物。傅里叶变换红外（FTIR）和核磁共振（NMR）光谱技术被用来确定结合到脂质体的离子的结构与模型集群的量子力学（QM）计算的帮助。QM和实验措施（ITC，FTIR，朗缪尔单层实验，和NMR）被用来parametrize添加剂和可极化的德鲁德力场的离子-水，-脂质和-蛋白质相互作用。这些力场将用于分子动力学（MD）模拟与增强采样方法，以研究钙（II）和铍（II）的协调和竞争性结合。该项目的最后一个方面是探测模型磷脂酰丝氨酸（PS）受体的脂质体和/或凋亡细胞的钙（II）和铍（II）的存在下，荧光显微镜，流式细胞术和电子顺磁共振的结合。在平行，钙（II）的铍（II）在钙（II）安排的PS接触这些蛋白质受体的位移进行了研究与MD模拟探测的协调和这些相互作用的强度的关键作用。这些研究有望为铍（II）如何干扰细胞内信号传导的机制提供深入了解，包括细胞外PS识别和Be（II）-破坏凋亡细胞清洁级联的细节。该奖项反映了NSF的法定使命，并被认为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来支持。

With this award, the Chemistry of Life Processes Program is funding Drs. Jeffery Klauda and Sergei Sukharev at the University of Maryland to study the binding competition of polyvalent ions with lipids and membrane-associated proteins. Calcium ions in solution play multiple critical roles as signaling agents and cofactors that mediate muscle contraction, nerve signal transmission, blood clotting, and, importantly, dead cell removal. Positively charged calcium ions assist in recognition of injured and dying cells by special biological processes, which depend on calcium binding to negatively-charged lipid molecules exposed during cell death. Specific proteins recognize these calcium-coated dying (apoptotic) cells and are key to removing these cells. However, some toxic metal ions, such as beryllium, can compete with, displace, and suppress natural functions mediated by calcium. Drs. Jeffery Klauda and Sergei Sukharev use a combined experimental and computational approach to understand the basic aspects of binding and competition of calcium and beryllium to model compounds, lipids, and proteins. This project provides insight into the mechanisms of how beryllium interferes with multiple calcium-dependent processes and specifically disrupts apoptotic cell recognition and the associated cleaning cascade in the body. This project provides training for undergraduate and graduate student researchers, and the research results are disseminated through presentations at scientific meetings and publications. This project involves educational outreach through summer research internships for high school students at minority-serving institutions. A series of magnet practicums will allow students to actively perform supervised research in the labs of Drs. Klauda and Sukharev. In addition, course lectures and practical examples from the project research are integrated into undergraduate and graduate engineering and science courses to increase engagement and exposure.Probing the structure and thermodynamics for ions binding to lipids in cellular membranes has been challenging. This project aims to use isothermal titration calorimetry (ITC) to obtain free energies of binding for calcium(II) and beryllium(II) to model compounds for lipids and peptide carbonyls/carboxylates. Fourier Transform-Infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopic techniques are used to determine the structure of ions binding to liposomes with the aid of quantum mechanical (QM) calculations of model clusters. QM and experimental measures (ITC, FTIR, Langmuir monolayer experiments, and NMR) are used to parametrize an additive and a polarizable Drude force field for ion-water, -lipid and -protein interactions. These force fields will be used in molecular dynamics (MD) simulations with enhanced sampling approaches to study the coordination and competitive binding of calcium(II) and beryllium(II). The final aspect of this project is probing the binding of model phosphatidylserine (PS) receptors to liposomes and/or apoptotic cells in the presence of calcium(II) and beryllium(II) with fluorescent microscopy, flow cytometry and electron paramagnetic resonance. In parallel, calcium(II) displacement by beryllium(II) in the calcium(II)-arranged PS contacts with these protein receptors is studied with MD simulations to probe the critical role of coordination and the strength of these interactions. Together these studies are expected to provide insight into the mechanism of how beryllium(II) disrupts intracellular signaling, including details of extracellular PS recognition and of Be(II)-disruption of the apoptotic cell cleaning cascade.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.