Department of Biomedical Engineering; Intercollege Graduate Degree Program in Bioengineering.
Department of Biomedical Engineering; Department of Physics, Pennsylvania State University, University Park, Pennsylvania.
Biophys J. 2019 Jul 23;117(2):331-345. doi: 10.1016/j.bpj.2019.06.010. Epub 2019 Jun 20.
High-resolution tracking of gold nanoparticle-labeled proteins has emerged as a powerful technique for measuring the structural kinetics of processive enzymes and other biomacromolecules. These techniques use point spread function (PSF) fitting methods borrowed from single-molecule fluorescence imaging to determine molecular positions below the diffraction limit. However, compared to fluorescence, gold nanoparticle tracking experiments are performed at significantly higher frame rates and utilize much larger probes. In the current work, we use Brownian dynamics simulations of nanoparticle-labeled proteins to investigate the regimes in which the fundamental assumptions of PSF fitting hold and where they begin to break down. We find that because gold nanoparticles undergo tethered diffusion around their anchor point, PSF fitting cannot be extended to arbitrarily fast frame rates. Instead, camera exposure times that allow the nanoparticle to fully populate its stationary positional distribution achieve a spatial averaging that increases fitting precision. We furthermore find that changes in the rotational freedom of the tagged protein can lead to artifactual translations in the fitted particle position. Finally, we apply these lessons to dissect a standing controversy in the kinesin field over the structure of a dimer in the ATP waiting state. Combining new experiments with simulations, we determine that the rear kinesin head in the ATP waiting state is unbound but not displaced from its previous microtubule binding site and that apparent differences in separately published reports were simply due to differences in the gold nanoparticle attachment position. Our results highlight the importance of gold conjugation decisions and imaging parameters to high-resolution tracking results and will serve as a useful guide for the design of future gold nanoparticle tracking experiments.
金纳米粒子标记蛋白的高分辨率追踪已成为测量连续酶和其他生物大分子结构动力学的强大技术。这些技术使用从单分子荧光成像借来的点扩散函数 (PSF) 拟合方法来确定低于衍射极限的分子位置。然而，与荧光相比，金纳米粒子跟踪实验在高得多的帧率下进行，并利用更大的探针。在目前的工作中，我们使用标记蛋白的纳米粒子的布朗动力学模拟来研究 PSF 拟合的基本假设成立的范围以及它们开始失效的范围。我们发现，由于金纳米粒子在其锚定点周围进行束缚扩散，PSF 拟合不能扩展到任意快的帧率。相反，允许纳米粒子完全填充其静止位置分布的相机曝光时间实现了增加拟合精度的空间平均。我们还发现，标记蛋白的旋转自由度的变化会导致拟合粒子位置出现人为的平移。最后，我们将这些教训应用于剖析在肌球蛋白领域中关于 ATP 等待状态中二聚体结构的一个长期争议。通过结合新的实验和模拟，我们确定 ATP 等待状态中的后向肌球蛋白头部未结合，但未从其先前的微管结合位点位移，并且分别发表的报告中的明显差异仅仅是由于金纳米粒子附着位置的差异。我们的结果强调了金缀合决策和成像参数对高分辨率跟踪结果的重要性，并将作为未来金纳米粒子跟踪实验设计的有用指南。
