Center for Neutron Research , National Institute of Standards and Technology , Gaithersburg , Maryland 20899 , United States.
Langmuir. 2019 Sep 17;35(37):12246-12256. doi: 10.1021/acs.langmuir.9b01928. Epub 2019 Sep 6.
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 , 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.
表面支撑的脂质双层在纳米科学领域被广泛用作细胞膜模拟物。例如，它们经常被用于单分子原子力显微镜（AFM）研究中，以揭示膜蛋白构象动力学和折叠。然而，在 AFM 以及其他表面感应技术中，支撑表面的近距离引发了对生物化学活性保存的质疑。在这里，我们使用一般分泌（Sec）系统的模型易位子，通过两种生化测定来定量研究表面支撑双层中的活性。第一种测定评估了 ATP 水解，第二种测定通过添加蛋白酶来防止多肽跨膜转运，从而评估了多肽的跨膜转运。水解测定显示了不同水平的激活，从中等（易位子激活）到高（转运相关），这与传统的溶液实验相似，并进一步鉴定了一种具有构象滞后特征的三磷酸腺苷酶群体。转运测定显示的周转率与溶液相当，但表观速率常数降低了 10 倍。尽管动力学存在差异，但与溶液相比，玻璃上的化学机械耦合（每转位残基水解的 ATP）仅变化两倍。活性随底层表面形貌复杂性而变化。粗糙的玻璃盖玻片比原子级平整的云母更受青睐，这可能是由于转运多肽与表面之间的摩擦耦合存在差异。中子反射测量和 AFM 证实了生化测量，并提供了亚膜空间和双层上表面的结构特征。总的来说，表面吸附的 Sec 系统的转运活性得以维持，尽管限速步骤较慢。更一般地说，多肽转运活性的测量提供了有价值的定量指标，可用于评估表面支撑脂质双层的局部环境。
