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

Abstract

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 2-3nm lateral and∼0.1nm vertical spatial resolution, and at sub-100ms 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.