Biozentrum, ‡Swiss Nanoscience Institute, and §Research IT, Biozentrum, University of Basel , Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
ACS Nano. 2017 Nov 28;11(11):10852-10859. doi: 10.1021/acsnano.7b04216. Epub 2017 Oct 17.
Multienzymes, such as the protein metazoan fatty acid synthase (FAS), are giant and highly dynamic molecular machines for critical biosynthetic processes. The molecular architecture of FAS was elucidated by static high-resolution crystallographic analysis, while electron microscopy revealed large-scale conformational variability in FAS with some correlation to functional states in catalysis. However, little is known about time scales of conformational dynamics, the trajectory of motions in individual FAS molecules, and the extent of coupling between catalysis and structural changes. Here, we present an experimental single-molecule approach to film immobilized or selectively tethered FAS in solution at different viewing angles and high spatiotemporal resolution using high-speed atomic force microscopy. Mobility of individual regions of the multienzyme is recognized in video sequences, and correlation of shape features implies a convergence of temporal resolution and velocity of FAS dynamics. Conformational variety can be identified and grouped by reference-free 2D class averaging, enabling the tracking of conformational transitions in movies. The approach presented here is suited for comprehensive studies of the dynamics of FAS and other multienzymes in aqueous solution at the single-molecule level.
多酶，如蛋白后生动物脂肪酸合酶（FAS），是用于关键生物合成过程的巨大的、高度动态的分子机器。FAS 的分子结构通过静态高分辨率晶体学分析得以阐明，而电子显微镜揭示了 FAS 的大规模构象变异性，与催化过程中的某些功能状态相关。然而，关于构象动力学的时间尺度、单个 FAS 分子中运动的轨迹以及催化和结构变化之间的耦合程度，我们知之甚少。在这里，我们提出了一种实验性的单分子方法，使用高速原子力显微镜在不同观察角度下以高时空分辨率在溶液中对固定或选择性固定的 FAS 进行成像。在视频序列中可以识别多酶的各个区域的运动性，并且形状特征的相关性表明了 FAS 动力学的时间分辨率和速度的收敛。通过无参考的 2D 类平均可以识别和分组构象多样性，从而能够在电影中跟踪构象转变。本文提出的方法适用于在水溶液中单分子水平上对 FAS 和其他多酶的动力学进行全面研究。
