CAREER: Plasticity, Promiscuity and Transport Mechanisms in Transmembrane Metal Pumps

职业：跨膜金属泵的可塑性、混杂性和传输机制

• 批准号: 2045984
• 负责人: Gabriele Meloni
• 金额: $ 67.79万
• 依托单位: University of Texas at Dallas
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2045984&HistoricalAwards=false
• 关键词: CAREER; Plasticity; Promiscuity; Transport; Mechanisms

With the support of the Chemistry of Life Processes Program in the Division of Chemistry, Dr. Gabriele Meloni from The University of Texas at Dallas will study the molecular mechanism of function of transmembrane transporter proteins involved in metal transport across cellular membranes. In all kingdoms of life, the activity of metal pumps guarantee that metal concentrations are regulated to meet cellular requirements. P-type ATPase pumps are a class of transmembrane proteins that utilize ATP as an energy source to move essential and toxic metals across membranes playing a central role in metal balance. However, the general molecular processes involved in specific metal cargo selection and transport remain to be revealed. Structural, biochemical, and biophysical methods will be applied to a comparative characterization of metal recognition in pump subclasses showing diverse metal selectivity patterns to address a new molecular level understanding of metal recognition and translocation. The expected results will contribute to a poorly explored area of biochemistry and will provide new insights regarding metal transport processes in living organisms. The research plan will be integrated with a multifaceted educational program focused on promoting interest and student engagement in bioinorganic chemistry. By developing new course content with peer-led delivery modalities, expanding undergraduate research engagement with a particular focus on underrepresented minorities, and creating a lab-based online video “reality-show” to be shared with Primarily Undergraduate Institutions (PUI) and high school students, the project will promote awareness, interest and societal knowledge in inorganic biochemistry.P1B-type ATPase are ATP-energized primary-active pumps that control translocation of both essential and toxic transition metals. The adaptation of a conserved topological and structural framework, common to all P-type ATPases, to the need of diverse substrate selectivity and promiscuity allowed the evolution of P1B-type subfamilies capable of selective translocation of first-row essential transition metals (Mn(II), Fe(II), Co(II), Ni(II), Cu(I) or Zn(II)), as well as toxic second- and third-row transition and post-transition metals (e.g. Ag(I), Cd(II), Hg(II), Pb(II)). The project will address, by in-house and synchrotron-based biophysical and biochemical methods, the atomic-level determinants controlling metal recognition, promiscuity and structural plasticity in poorly characterized metal pumps subclasses (P1B-4-P1B-7 types) with diverse selectivity patterns. These investigations will be complemented with real-time fluorescence measurements of metal transport events in proteoliposomes combined with the use of fluorescent sensors to investigate putative co-transported ions and membrane potential generation, to provide highlights on the overall kinetics, thermodynamics and mechanism of transport. This integrated approach will reveal new paradigms underlying energy-dependent selective metal translocation processes and define how plasticity and promiscuous recognition is exploited to guarantee metal translocation across membranes.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，来自德克萨斯大学达拉斯分校的Gabriele Meloni博士将研究跨膜转运蛋白功能的分子机制，这些蛋白参与金属跨细胞膜的转运。在所有的生命王国中，金属泵的活动保证了金属浓度的调节，以满足细胞的需求。P型ATP酶泵是一类跨膜蛋白，其利用ATP作为能量来源来跨膜移动必需和有毒金属，在金属平衡中起核心作用。 然而，特定金属货物的选择和运输所涉及的一般分子过程仍有待揭示。结构，生物化学和生物物理的方法将被应用到一个比较表征的金属识别泵子类显示不同的金属选择性模式，以解决一个新的分子水平的理解金属识别和易位。预期的结果将有助于生物化学的一个探索不足的领域，并将提供新的见解，在活生物体中的金属运输过程。该研究计划将与多方面的教育计划相结合，重点是促进学生对生物无机化学的兴趣和参与。该项目通过开发新的课程内容，以同伴为主导的交付模式，扩大本科生的研究参与，特别关注代表性不足的少数群体，并创建一个基于实验室的在线视频“真人秀”，与小学本科院校（PUI）和高中学生分享，将提高认识，对无机生物化学兴趣和社会知识。2 P1 B型ATP酶是ATP供能的初级活性泵，控制必需的和有毒的过渡金属的移位。所有P-型ATP酶共有的保守拓扑和结构框架适应于不同底物选择性和混杂性的需要，允许P1 B-型亚家族的进化，所述P1 B-型亚家族能够选择性转运第一行必需过渡金属（Mn（II）、Fe（II）、Co（II）、Ni（II）、Cu（I）或Zn（II）），以及有毒的第二和第三行过渡金属和后过渡金属（如Ag（I）、Cd（II）、Hg（II）、Pb（II））。该项目将通过内部和基于同步加速器的生物物理和生物化学方法，解决控制具有不同选择性模式的特征不佳的金属泵子类（P1 B-4-P1 B-7类型）中的金属识别，混杂和结构可塑性的原子级决定因素。这些调查将补充与实时荧光测量的金属运输事件的蛋白脂质体结合使用荧光传感器，以调查推定的共运输离子和膜电位的产生，提供重点的整体动力学，热力学和运输机制。这一综合方法将揭示新的范例潜在的能源依赖性选择性金属易位过程和定义如何利用可塑性和混杂识别，以保证金属易位跨membranes.This奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查标准的支持。