Abstract：
Transmembrane transition metal transporter proteins are central gatekeepers in selectively controlling vectorial metal cargo uptake and extrusion across cellular membranes in all living organisms, thus playing key roles in essential and toxic metal homeostasis. Biochemical characterization of transporter-mediated translocation events and transport kinetics of redox-active metals, such as iron and copper, is challenged by the complexity in generating reconstituted systems in which vectorial metal transport can be studied in real time. We present fluorescence-based proteoliposome methods to monitor redox-active metal transmembrane translocation upon reconstitution of purified metal transporters in artificial lipid bilayers. By encapsulating turn-on/-off iron or copper-dependent sensors in the proteoliposome lumen and conducting real-time transport assays using small unilamellar vesicles (SUVs), in which selected purified Fe(II) and Cu(I) transmembrane importer and exporter proteins have been reconstituted, we provide a platform to monitor metal translocation events across lipid bilayers in real time. The strategy is modular and expandable toward the study of different transporter families featuring diverse metal substrate selectivity and promiscuity.
摘要：
跨膜过渡金属转运蛋白是在所有活生物体中选择性控制载体金属货物在细胞膜上的摄取和挤出的中心看门人。从而在必需和有毒金属稳态中起关键作用。转运蛋白介导的易位事件的生化表征和氧化还原活性金属的转运动力学，如铁和铜，由于生成重组系统的复杂性而面临挑战，在该系统中可以实时研究矢量金属传输。我们提出了基于荧光的蛋白脂质体方法，以监测人工脂质双层中纯化的金属转运蛋白重建后的氧化还原活性金属跨膜易位。通过在蛋白脂质体腔中封装开启/关闭铁或铜依赖性传感器，并使用小型单层囊泡（SUV）进行实时转运测定，其中选定的纯化的Fe（II）和Cu（I）跨膜转运蛋白和转运蛋白已被重建，我们提供了一个平台来实时监测跨脂双层的金属易位事件.该策略是模块化的，可扩展用于研究具有多种金属底物选择性和混杂性的不同转运蛋白家族。
