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

在化学系生命过程化学项目的支持下，德克萨斯大学达拉斯分校的加布里埃尔·梅洛尼博士将研究跨膜转运蛋白参与金属跨细胞膜运输的分子机制。在所有生命王国中，金属泵的活动保证了金属浓度受到调节，以满足细胞的要求。P型ATPase泵是一类跨膜蛋白，它利用三磷酸腺苷作为能量源，将必需的和有毒的金属跨膜运输，在金属平衡中起着核心作用。然而，涉及特定金属货物选择和运输的一般分子过程仍有待揭示。结构、生化和生物物理方法将被应用于泵亚类中金属识别的比较表征，显示出不同的金属选择性模式，以解决对金属识别和移位的新的分子水平的理解。预期的结果将有助于生物化学中一个尚未被探索的领域，并将为生物体内的金属运输过程提供新的见解。该研究计划将与一个多方面的教育计划相结合，重点是提高学生对生物无机化学的兴趣和参与度。通过开发新的课程内容，采用同行主导的教学模式，扩大本科生的研究参与，特别关注未被充分代表的少数群体，并创建一个以实验室为基础的在线视频“真人秀”，主要与本科院校(PUI)和高中生分享，该项目将提高人们对无机生物化学的认识、兴趣和社会知识。P1B型ATPase是由ATP提供能量的初级活性泵，控制必需和有毒过渡金属的转移。所有P型ATPase共有的保守的拓扑和结构框架的适应，以适应不同底物选择性和混杂的需要，允许P1B型亚家族的进化，能够选择性地移位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(例如，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化方法，研究控制金属识别、混杂和结构可塑性的原子级决定因素，这些决定因素在具有不同选择模式的特征较差的金属泵亚类(P1B-4-P1B-7型)中起决定作用。这些研究将辅之以实时荧光测量蛋白脂质体中的金属转运事件，并结合使用荧光传感器来研究假定的共转运离子和膜电位产生，以提供关于整体动力学、热力学和转运机制的重点。这一综合方法将揭示基于能量的选择性金属转移过程的新范式，并定义如何利用可塑性和混杂识别来保证金属跨膜转移。该奖项反映了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, 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.