Protein Translocation Activity in Surface-Supported Lipid Bilayers
Protein Translocation Activity in Surface-Supported Lipid Bilayers
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DOI：
10.1021/acs.langmuir.9b01928
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发表时间：
2019-09-17
期刊：
影响因子：
3.9
通讯作者：
King, Gavin M.
中科院分区：
文献类型：
--
作者：
Chattrakun, Kanokporn;Hoogerheide, David P.;King, Gavin M.
表面支撑的脂质双层作为细胞膜模拟物在整个纳米科学界广泛使用。例如，它们经常用于单分子原子力显微镜（AFM）研究，以揭示膜蛋白的构象动力学和折叠。然而，在AFM以及其他表面传感技术中，支撑表面的紧密接近引起了关于生物化学活性保存的问题。从大肠杆菌的一般分泌（Sec）系统的模型易位酶，在这里，我们通过两个生化测定在表面支持的双层定量的活动。第一个评估ATP水解，第二个评估通过保护免受添加的蛋白酶的多肽跨膜易位。水解试验揭示了不同水平的激活范围从中等（易位激活）到高（易位相关），这是类似于传统的解决方案的实验，并进一步确定了一个腺苷三磷酸酶人口表现出的构象滞后的特点。易位试验显示，周转数与溶液相当，但表观速率常数降低了10倍。尽管动力学的差异，化学机械耦合（ATP水解每个残基易位）只有两个不同的玻璃相比，解决方案。下垫面的地形复杂程度不同，其活动性也不同。粗糙的玻璃盖玻片比原子级平坦的云母更受青睐，这可能是由于易位多肽和表面之间的摩擦耦合的差异。中子反射仪和原子力显微镜证实了生化测量，并提供了结构表征的亚膜空间和上表面的双层。总体而言，易位活性保持表面吸附的Sec系统，虽然与一个较慢的限速步骤。更一般地，多肽易位活性测量产生有价值的定量指标，以评估局部环境的表面支持的脂质双层。
Surface-supported lipid bilayers are used widely throughout the nanoscience community as cellular membrane mimics. For example, they are frequently employed in single-molecule atomic force microscopy (AFM) studies to shed light on membrane protein conformational dynamics and folding. However, in AFM as well as in other surface-sensing techniques, the close proximity of the supporting surface raises questions about preservation of the biochemical activity. Employing the model translocase from the general secretory (Sec) system of Escherichia coli, here we quantify the activity via two biochemical assays in surface supported bilayers. The first assesses ATP hydrolysis and the second assesses polypeptide translocation across the membrane via protection from added protease. Hydrolysis assays revealed distinct levels of activation ranging from medium (translocase-activated) to high (translocation-associated) that were similar to traditional solution experiments and further identified an adenosine triphosphatase population exhibiting characteristics of conformational hysteresis. Translocation assays revealed turn over numbers that were comparable to solution but with a 10-fold reduction in apparent rate constant. Despite differences in kinetics, the chemomechanical coupling (ATP hydrolyzed per residue translocated) only varied twofold on glass compared to solution. The activity changed with the topographic complexity of the underlying surface. Rough glass coverslips were favored over atomically flat mica, likely due to differences in frictional coupling between the translocating polypeptide and surface. Neutron reflectometry and AFM corroborated the biochemical measurements and provided structural characterization of the submembrane space and upper surface of the bilayer. Overall, the translocation activity was maintained for the surface-adsorbed Sec system, albeit with a slower rate-limiting step. More generally, polypeptide translocation activity measurements yield valuable quantitative metrics to assess the local environment about surface-supported lipid bilayers.