Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158.
Howard Hughes Medical Institute, University of California, San Francisco, CA 94158.
Proc Natl Acad Sci U S A. 2019 Mar 5;116(10):4275-4284. doi: 10.1073/pnas.1815826116. Epub 2019 Feb 15.
Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil "stalk" of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.
光学显微镜是一种强大的工具，可用于探测单分子水平上分子机器的构象。单分子Förster 共振能量转移可测量单分子在 2 至 8nm 范围内的分子内距离变化。然而，目前的超分辨率测量在低于 25nm 时变得容易出错。因此，需要新的单分子方法来测量 8 至 25nm 范围内的距离。在这里，我们描述了利用定位和成像误差信息来测量两个不同颜色荧光团之间距离的方法，在 >2nm 的距离处具有约 1nm 的精度。这些技术可以使用标准的全内反射荧光显微镜和开源软件进行高通量实现。我们将我们的双色定位方法应用于揭示动力蛋白 dynein 卷曲“茎”中的一个出乎意料的约 4nm 核苷酸依赖性构象变化。我们预计这些方法将有助于在广泛的长度范围内对单分子进行高精度的距离测量。
