Szathmáry Zsófia Edit, Pedersen Martin Cramer, Michels Alec, Regueira Torsten Høybye Bak, Kirkensgaard Jacob Judas Kain
Department of Food Science, University of Copenhagen, Rolighedsvej 26, 1958, Frederiksberg, Denmark.
Aquaporin A/S, Nymøllevej 78, 2800, Kgs. Lyngby, Denmark.
Eur Biophys J. 2025 Aug 7. doi: 10.1007/s00249-025-01790-8.
Aquaporins are known for their efficient water transport capabilities and have been widely studied in the past decades. However, creating a biomimetic system mirroring natural water filtration processes still poses a challenge related to performance and stability. To study the protein reconstitution and functionality, this work presents an analytical toolkit using the model system of AqpZ reconstituted phosphatidylcholine proteoliposomes. Combining findings from dynamic light scattering, cryogenic transmission electron microscopy, laser scanning confocal microscopy, stimulated emission depletion microscopy, stopped flow-light scattering and small-angle X-ray scattering provides an assessment of structural and functional characteristics of AqpZ embedding in the bilayer of liposomes. Findings of this work reveal that the incorporation of AqpZ into liposomes promotes an increase within the hydrophobic bilayer thickness as well as within the overall size of the vesicles. AqpZ, AqpZ-GFP and AqpZ-Atto594 are studied and show distinct permeability profiles. Despite all three displaying a successful structural reconstitution into the liposomes, labeled protein variants demonstrate a loss of function. A series of protein concentrations are utilized to extract quantitative information regarding the reconstitution process, revealing constant water transport per AqpZ and thus a consistent trend of increased reconstitution and permeability as a function of AqpZ concentration, as determined by stopped flow-light scattering and detailed further via global fitting of small-angle X-ray scattering data.
水通道蛋白以其高效的水运输能力而闻名，在过去几十年中得到了广泛研究。然而，创建一个模仿天然水过滤过程的仿生系统在性能和稳定性方面仍然面临挑战。为了研究蛋白质的重构和功能，这项工作提出了一种分析工具包，该工具包使用了AqpZ重构的磷脂酰胆碱蛋白脂质体模型系统。结合动态光散射、低温透射电子显微镜、激光扫描共聚焦显微镜、受激发射损耗显微镜、停流-光散射和小角X射线散射的研究结果，可以评估AqpZ嵌入脂质体双层中的结构和功能特征。这项工作的研究结果表明，将AqpZ掺入脂质体中会导致疏水双层厚度以及囊泡整体尺寸增加。对AqpZ、AqpZ-GFP和AqpZ-Atto594进行了研究，结果显示出不同的渗透特性。尽管这三种蛋白都成功地重构到了脂质体中，但标记的蛋白质变体却表现出功能丧失。利用一系列蛋白质浓度来提取有关重构过程的定量信息，结果表明每个AqpZ的水运输量恒定，因此重构和渗透率随AqpZ浓度增加的趋势一致，这是通过停流-光散射确定的，并通过小角X射线散射数据的全局拟合进一步详细分析得出的。
