Younus Iqra, Ford Robert C, Prince Stephen M
Faculty of Biology, Medicine and Health, School of Biological Sciences, The University of Manchester, Manchester M13 9PT, UK.
Membranes (Basel). 2025 Jan 10;15(1):20. doi: 10.3390/membranes15010020.
Adenosine triphosphate-binding cassette (ABC) transporters form a ubiquitous superfamily of integral membrane proteins involved in the translocation of substrates across membranes. Human ABC transporters are closely linked to the pathogenesis of diseases such as cancer, metabolic diseases, and Alzheimer's disease. In this study, four ABC transporters were chosen based on (I) their importance in humans and (II) their score in a structural bioinformatics screen aimed at the prediction of crystallisation propensity. The top-scoring ABC transporters' orthologs (-mouse ABCB5, -giant panda ABCB6, -little brown bat ABCG1 and ABCG4) were then expressed in with a combined green fluorescent protein and polyhistidine tag, enabling visualisation and purification. After partial purification and in the presence of the detergent (n-dodecyl-β-D-maltoside), the kinetic parameters of the ATP hydrolysis reactions of the orthologs were determined, as well as the extent of stimulation of their activity when presented with putative substrates. We discuss the efficiency of such bioinformatics approaches and make suggestions for their improvement and wider application in membrane protein-structure determination.
三磷酸腺苷结合盒（ABC）转运蛋白构成了一个普遍存在的整合膜蛋白超家族，参与底物跨膜转运。人类ABC转运蛋白与癌症、代谢性疾病和阿尔茨海默病等疾病的发病机制密切相关。在本研究中，基于以下两点选择了四种ABC转运蛋白：（I）它们在人类中的重要性；（II）它们在旨在预测结晶倾向的结构生物信息学筛选中的得分。然后，得分最高的ABC转运蛋白的直系同源物（-小鼠ABCB5、-大熊猫ABCB6、-小棕蝠ABCG1和ABCG4）与绿色荧光蛋白和多聚组氨酸标签一起表达，以便进行可视化和纯化。在部分纯化后，在去污剂（正十二烷基-β-D-麦芽糖苷）存在的情况下，测定了直系同源物ATP水解反应的动力学参数，以及在提供推定底物时其活性的刺激程度。我们讨论了此类生物信息学方法的效率，并对其在膜蛋白结构测定中的改进和更广泛应用提出了建议。
