Resolution of angstrom-scale protein-conformational changes by analyzing fluorescence anisotropy

Conformational changes within typical protein molecules are rapid and small, making their quantitative resolution challenging. These changes generally involve rotational motions, and may thus be resolved by determining changes in the orientation of a fluorescent label that assumes a unique orientation in each conformation. Here, by analyzing fluorescence intensities collected using a polarization microscope at a rate of 50 frames per second, we can follow the changes of 10 to 16° in the orientation of a single bifunctional rhodamine molecule attached to an RCK domain of the MthK channel, and thus the transitions among its three conformational states, with effective standard deviation (σ) of 2 to 5°. Based on available crystal structures, the position of the fluorophore's center differ by 3.4 to 8.1 Å among the states. Thus, the present approach allows the resolution of protein-conformational changes involving angstrom-scale displacements.