Structural and Functional Analyses of IDPs by High-Speed AFM Imaging

Abstract

The structure of intrinsically disordered proteins (IDPs) is highly flexible and dynamically samples a multitude of conformational states. Therefore, the structural characterization of IDPs is considerably challenging. Here, we developed a single-molecule method to analyze their structure, dynamics and function, using high-speed atomic force microscopy (HS-AFM). A mechanical property of constantly or fully disordered regions within IDPs was found to be independent of their amino-acid sequences. Thanks to the invariance of this property, HS-AFM imaging of IDPs allowed us to estimate the number of amino acids contained in these regions as well as to identify and characterize at the residue level other regions undergoing order-to-disorder transitions. As a test sample for functional visualization by HS-AFM, we imaged the methyl-CpG-binding protein MeCP2, whose gene mutations cause neurodevelopmental disorder called Rett syndrome. Its methyl-CpG binding domain (MBD) was observed to undergo order-to-disorder transitions, whereas other regions were mostly or constantly disordered. In MBDs of several mutated MeCP2, this two-state equilibrium was shifted to the disordered state with different degrees. This disorder propensity was found to be tightly correlated to the lower activity of MeCP2 to condense methylated dsDNA, and hence, their lower activity to condense DNA must be directly involved in causing Rett syndrome. Thus, HS-AFM imaging of single molecules opens a new opportunity to readily acquire site-resolved, quantitative and dynamic structural information on IDPs in real space as well as to study how minute changes in the structure and dynamics of IDPs are connected to diseases.