Diffracted X-ray tracking method for recording single-molecule protein motions

Background: Proteins change their conformation depending on function. Although a vast number of static pictures of proteins have been accumulated, information regarding their dynamics in function is limited. Diffracted X-ray tracking (DXT) is a good candidate to obtain the missing data. Scope of review: A gold nanocrystal was attached to the target protein as a probe and the motion of the X-ray diffraction spots from the crystal corresponded to the motion of the target. Although it has advantages of high temporal (sub-millisecond) and spatial (approximately 0.1 degrees) resolutions, it is not extensively utilized. This review focused on its effective application from a user's perspective. We also present an example with the KcsA channel and the status of recent developments to show the future possibilities of the method. Major conclusions: DXT is a powerful method to investigate intramolecular structural changes. For instance, in the KcsA channel, the method revealed a wave of conformational changes transmitted from the gate region to the end of the molecule. The method is continuously being developed, and users can choose an appropriate measurement system depending on the condition of their sample. General significance: Revealing the protein structural changes with respect to function is an important frontier. The most distinctive feature of the DXT method is that both high temporal and spatial resolutions are achievable, and it is possible to track the motions of multiple molecules at the same time. This feature is an advantage for screening molecules associated with the target proteins (e.g., ligands and medicines).