Hatty Claire R, Le Brun Anton P, Lake Vanessa, Clifton Luke A, Liu Guo Jun, James Michael, Banati Richard B
Medical Imaging & Radiation Sciences Faculty Research Group, Faculty of Health Sciences, The University of Sydney, c/o Brain & Mind Research Institute, 94 Mallett Street, Camperdown, NSW 2050, Australia.
Bragg Institute, Australian Nuclear Science and Technology Organisation, New Illawarra Road, Lucas Heights, NSW 2234, Australia.
Biochim Biophys Acta. 2014 Mar;1838(3):1019-30. doi: 10.1016/j.bbamem.2013.12.013. Epub 2013 Dec 27.
The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug-membrane interactions may contribute to the apparent affinity of TSPO ligands.
药物配体的功能效应可能不仅归因于其与膜蛋白靶点的相互作用，还与其周围的脂质膜有关。我们使用朗缪尔单层膜、具有耗散监测功能的石英晶体微天平（QCM-D）和中子反射率（NR）研究了药物配体PK11195与其主要蛋白靶点——整合膜18 kDa转位蛋白（TSPO）以及模型膜之间的相互作用。我们发现PK11195可掺入脂质单层膜和脂质双层膜中，导致脂质分子面积减小以及脂质双层膜刚性增加。NR显示PK11195以约10%的体积分数掺入脂质链区域。我们将分离的小鼠TSPO重组到脂质双层膜中，并使用QCM-D研究其与PK11195的相互作用，结果显示频率响应大于预期，表明该蛋白可能发生了构象变化。NR测量结果显示，通过其多组氨酸标签固定在修饰表面上时，TSPO的表面覆盖率为23%，TSPO层的厚度为51Å。这些技术使我们能够探究TSPO与PK11195之间的相互作用以及PK11195与模型膜之间的相互作用。之前报道的PK11195不依赖于TSPO的效应可能是由于其掺入脂质双层膜并改变了其物理性质。这也对观察到的TSPO配体的可变结合谱有影响，因为药物-膜相互作用可能有助于TSPO配体的表观亲和力。
