Guide to studying intrinsically disordered proteins by high-speed atomic force microscopy

Intrinsically disordered proteins (IDPs) are partially or entirely disordered. Their intrinsically disordered regions (IDRs) dynamically explore a wide range of structural space by their highly flexible nature. Due to this distinct feature largely different from structured proteins, conventional structural analyses relying on ensemble averaging is unsuitable for characterizing the dynamic structure of IDPs. Therefore, single-molecule measurement tools have been desired in IDP studies. High-speed atomic force microscopy (HS-AFM) is a unique tool that allows us to directly visualize single biomolecules at 2-3 nm lateral and similar to 0.1 nm vertical spatial resolution, and at sub-100 ms temporal resolution under near physiological conditions, without any chemical labeling. HS-AFM has been successfully used not only to characterize the shape and motion of IDP molecules but also to visualize their function-related dynamics. In this article, after reviewing the principle and current performances of HS-AFM, we describe experimental considerations in the HS-AFM imaging of IDPs and methods to quantify molecular features from captured images. Finally, we outline recent HS-AFM imaging studies of IDPs.

Automated summary

Intrinsically disordered proteins (IDPs) are proteins that are partially or entirely disordered, and their intrinsically disordered regions (IDRs) explore a wide range of structural space due to their highly flexible nature. Conventional structural analyses are not suitable for studying the dynamic structure of IDPs, so single-molecule measurement tools are needed. High-speed atomic force microscopy (HS-AFM) is a unique tool that allows direct visualization of the shape and motion of individual biomolecules under near physiological conditions, without any chemical labeling.