Nanometer-accuracy distance measurements between fluorophores at the single-molecule level
Open Access
- 15 February 2019
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 116 (10), 4275-4284
- https://doi.org/10.1073/pnas.1815826116
Abstract
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.Keywords
Funding Information
- HHS | NIH | National Institute of Biomedical Imaging and Bioengineering (R01EB007187)
- HHS | NIH | National Institute of General Medical Sciences (R35GM118106)
- HHS | NIH | NIH Office of the Director (1F32GM113366)
- HHS | NIH | National Institute of General Medical Sciences (R00GM112982)
- Damon Runyon Cancer Research Foundation (DFS-20-16)
- Howard Hughes Medical Institute (Investigator)
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