University of Tübingen, Institute of Applied Physics, Auf der Morgenstelle 10, 72076, Tübingen, Germany; Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya 1a, 119435, Moscow, Russia; Lomonosov Moscow State University, Faculty of Physics, Leninskie Gory 1/2, 119991, Moscow, Russia; National University of Science and Technology MISIS, Leninskiy prospekt 4, 119049, Moscow, Russia.
Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya 1a, 119435, Moscow, Russia; Moscow Institute of Physics and Technology (State University), Institutskiy per. 9, 141701, Dolgoprudny, Moscow region, Russia.
Colloids Surf B Biointerfaces. 2020 Sep;193:111077. doi: 10.1016/j.colsurfb.2020.111077. Epub 2020 Apr 27.
Understanding protein unfolding on a surface is of vital importance in nanoscience, nanobiotechnology, and medicine. Surfaces that retain the native conformation of adsorbed proteins (antimetamorphic surfaces) represent one of the main strategies for creating biocompatible materials, which are in great demand in biotechnology. Though the influence of surfaces on protein conformation has been studied for decades, real-time investigations of protein conformational behavior on a surface obtained at the single-molecule or sub-molecular level are still lacking and remain a challenge. In this work, we apply time-lapse atomic force microscopy (AFM) in aqueous solution to visualize the conformational dynamics of individual model protein molecules (E.coli RNA polymerase, RNAP) adsorbed on modified highly oriented pyrolytic graphite (HOPG) surfaces. We quantitatively characterize the evolution of height and shape of individual RNAP molecules adsorbed on a HOPG surface modified with an oligoglycine-hydrocarbon graphite modifier (GM) during the unfolding process and determine the characteristic unfolding time as ∼7 min. Furthermore, we make a HOPG surface antimetamorphic by modifying it with a denatured RNAP protein layer. Our results provide direct evidence of GM-HOPG-induced RNAP unfolding at the single-molecule level and open new strategies for the development and investigation of antimetamorphic graphitic surfaces.
了解表面上的蛋白质展开对于纳米科学、纳米生物技术和医学至关重要。保留吸附蛋白质的天然构象的表面（反变态表面）是创造生物相容性材料的主要策略之一，而生物相容性材料在生物技术中有很大的需求。尽管几十年来一直在研究表面对蛋白质构象的影响，但在单分子或亚分子水平上实时研究蛋白质在表面上的构象行为仍然缺乏，这仍然是一个挑战。在这项工作中，我们在水溶液中应用时间分辨原子力显微镜 (AFM) 来可视化单个模型蛋白质分子（大肠杆菌 RNA 聚合酶，RNAP）在经过修饰的高取向热解石墨 (HOPG) 表面上的构象动力学。我们定量地表征了在寡甘氨酸-碳氢化合物石墨修饰剂 (GM) 修饰的 HOPG 表面上吸附的单个 RNAP 分子在展开过程中高度和形状的演变，并确定了特征展开时间约为 7 分钟。此外，我们通过用变性的 RNAP 蛋白质层修饰 HOPG 表面使其反变态。我们的结果提供了 GM-HOPG 诱导的 RNAP 在单分子水平上展开的直接证据，并为开发和研究反变态石墨表面开辟了新的策略。
