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.
Highlights
Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level
To achieve highly accurate distance measurements between two fluorophores that emit at different wavelengths, multiple obstacles have to be overcome
We have developed nanometeraccuracy distance measurements for two differently colored fluorophores bound to static proteins
Summary
Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiledcoil “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. We applied our methods to investigate nucleotidedependent conformational changes of the molecular motor dynein [10,11,12] and found that the stalk of dynein likely undergoes a large conformational change during its hydrolysis cycle [13] These results could not have been obtained by smFRET or other direct two-color imaging methods, since the distances measured changed from ∼16 nm to ∼20 nm in different nucleotide states. We report methods capable of reliably measuring twocolor fluorophore distances at ∼1-nm accuracy over a wide range
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