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A single-liposome assay that enables temperature-dependent measurement of proton permeability of extremophile-inspired lipid membranes
Biochimica et Biophysica Acta (BBA) - Biomembranes ( IF 2.8 ) Pub Date : 2021-01-18 , DOI: 10.1016/j.bbamem.2021.183567
Anirvan Guha 1 , Melissa L McGuire 1 , Geoffray Leriche 2 , Jerry Yang 2 , Michael Mayer 1
Affiliation  

Thermoacidophilic archaea are able to survive in low pH environments by maintaining large proton gradients across their cell membranes. The unique structure of the unipolar and bipolar lipids that compose these membranes, with branched isoprene chains and ether linkages, may contribute to their reduced permeability to protons at elevated temperature. Here we present a proton permeability assay that interrogates hundreds of surface-tethered single liposomes in parallel using total internal reflection fluorescence (TIRF) microscopy to determine a proton permeability value for each liposome. The large number of liposomes that can be individually interrogated by this microscopy platform allow for the collection and statistical analysis of large quantities of data within a single experiment, as opposed to the single, averaged result produced by a bulk fluorimetry assay. In addition, tethering individual liposomes to a surface prevents liposome aggregation in free solution, which is especially prevalent when performing studies at elevated temperatures. Using a temperature-controlled chamber, we demonstrated a decreased temperature-dependence of proton permeability for bipolar archaea-mimetic membranes in comparison to unipolar eukaryote-mimetic membranes. Eyring-Polanyi analysis revealed a high entropic cost associated with proton flux across the archaea-mimetic membranes. Furthermore, analysis of the distribution of proton permeability values within populations of liposomes revealed a positive correlation between size and permeability in the eukaryote-mimetic liposomes that was absent in the archaea-mimetic liposomes.



中文翻译:

一种单脂质体测定法,可实现温度依赖性的极端微生物激发的脂膜质子渗透性的测量

嗜热古细菌能够在整个细胞膜上维持较大的质子梯度,从而能够在低pH环境中生存。组成这些膜的单极性和双极性脂质的独特结构,具有支链异戊二烯链和醚键,可能会导致它们在高温下对质子的渗透性降低。在这里,我们提出了一种质子渗透率测定法,该质子渗透率测定法使用全内反射荧光(TIRF)显微镜并行地查询数百个表面束缚的单个脂质体,以确定每个脂质体的质子渗透率值。可以通过此显微镜平台单独查询的大量脂质体可以在单个实验(而不是单个实验)中收集和统计分析大量数据 体荧光测定法产生的平均结果。另外,将单个脂质体束缚在表面上可防止脂质体在游离溶液中聚集,这在高温下进行研究时尤为普遍。使用温度控制室,我们证明了与单极真核生物模拟膜相比,双极古细菌模拟膜的质子渗透性的温度依赖性降低。Eyring-Polanyi分析表明,与穿过古细菌模拟膜的质子通量相关的熵成本很高。此外,对脂质体群体内质子渗透性值的分布的分析表明,真核生物模拟脂质体中缺少的真核生物模拟脂质体的大小与渗透性之间呈正相关。将单个脂质体束缚在表面上可防止脂质体在游离溶液中聚集,这在高温下进行研究时尤为普遍。使用温度控制室,我们证明了与单极真核生物模拟膜相比,双极古细菌模拟膜的质子渗透性的温度依赖性降低。Eyring-Polanyi分析表明,与穿过古细菌模拟膜的质子通量相关的熵成本很高。此外,对脂质体群体内质子渗透性值的分布的分析表明,真核生物模拟脂质体中缺少的真核生物模拟脂质体的大小与渗透性之间呈正相关。将单个脂质体束缚在表面上可防止脂质体在游离溶液中聚集,这在高温下进行研究时尤为普遍。使用温度控制室,我们证明了与单极真核生物模拟膜相比,双极古细菌模拟膜的质子渗透性的温度依赖性降低。Eyring-Polanyi分析表明,与穿过古细菌模拟膜的质子通量相关的熵成本很高。此外,对脂质体群体内质子渗透性值的分布的分析表明,真核生物模拟脂质体中缺少的真核生物模拟脂质体的大小与渗透性之间呈正相关。在高温下进行研究时尤为普遍。使用温度控制室,我们证明了与单极真核生物模拟膜相比,双极古细菌模拟膜的质子渗透性的温度依赖性降低。Eyring-Polanyi分析表明,与穿过古细菌模拟膜的质子通量相关的熵成本很高。此外,对脂质体群体内质子渗透性值的分布的分析表明,真核生物模拟脂质体中缺少的真核生物模拟脂质体的大小与渗透性之间呈正相关。在高温下进行研究时尤为普遍。使用温度控制室,我们证明了与单极真核生物模拟膜相比,双极古细菌模拟膜的质子渗透性的温度依赖性降低。Eyring-Polanyi分析表明,与穿过古细菌模拟膜的质子通量相关的熵成本很高。此外,对脂质体群体内质子渗透性值的分布的分析表明,真核生物模拟脂质体中缺少的真核生物模拟脂质体的大小与渗透性之间呈正相关。我们证明了与单极真核生物模拟膜相比,双极古细菌模拟膜的质子渗透性的温度依赖性降低。Eyring-Polanyi分析表明,与穿过古细菌模拟膜的质子通量相关的熵成本很高。此外,对脂质体群体内质子渗透性值的分布的分析表明,真核生物模拟脂质体中缺少的真核生物模拟脂质体的大小与渗透性之间呈正相关。我们证明了与单极真核生物模拟膜相比,双极古细菌模拟膜的质子渗透性的温度依赖性降低。Eyring-Polanyi分析表明,与穿过古细菌模拟膜的质子通量相关的熵成本很高。此外,对脂质体群体内质子渗透性值的分布的分析表明,真核生物模拟脂质体中缺少的真核生物模拟脂质体的大小与渗透性之间呈正相关。

更新日期:2021-01-24
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