High accuracy measurements of nanometer-scale distances between fluorophores at the single-molecule level

Abstract

To uncover the mechanisms of molecular machines it is useful to probe their structural conformations. Single-molecule Forster resonance energy transfer (smFRET) is a powerful tool for measuring intra-molecular shape changes of single-molecules, but is confined to distances of 2-8 nm. Current super-resolution measurements are error prone at 2 nm, using a standard TIRF microscope and open-source software. We applied our two-color localization method to uncover a [~]4 nm conformational change in the \"stalk\" of the motor protein dynein, revealing unexpected flexibility in this antiparallel coiled-coil domain. These new methods enable high-accuracy distance measurements of single-molecules that can be used over a wide range of length scales.