Visualization of Intrinsically Disordered Regions of Proteins by High‐Speed Atomic Force Microscopy

Abstract

Intrinsically disordered (ID) regions of proteins are recognized to be involved in biological processes such as transcription, translation, and cellular signal transduction. Despite the important roles of ID regions, effective methods to observe these thin and flexible structures directly were not available. Herein, we use high-speed atomic force microscopy (AFM) to observe the heterodimeric FACT (facilitates chromatin transcription) protein, which is predicted to have large ID regions in each subunit. Successive AFM images of FACT on a mica surface, captured at rates of 5-17 frames per second, clearly reveal two distinct tail-like segments that protrude from the main body of FACT and fluctuate in position. Using deletion mutants of FACT, we identify these tail segments as the two major ID regions predicted from the amino acid sequences. Their mechanical properties estimated from the AFM images suggest that they have more relaxed structures than random coils. These observations demonstrate that this state-of-the-art microscopy method can be used to characterize unstructured protein segments that are difficult to visualize with other experimental techniques.

Imaging stability in force-feedback high-speed atomic force microscopy

“…The striation pattern has been observed previously and found to be due to the resonant vibration of the piezo tube scanner (e.g. [20][21][22][23][24]). Therefore, the dominant factor that limits the response speed of the system at 0.1 s per frame was the resonance of the piezo tube [14].…”

High-speed atomic force microscopy coming of age

“…Imaging rate, 1.03 fps (×20 playback); scan size, 800 × 800 nm 2 [98]. ・Crystallization of a CaCO 3 thin film from supersaturated solution [72] ・Ultrafast imaging of collagen [58] ・Brownian motion & photo-degradation of π-conjugated polyrotaxane [73] ・DNA translocation and looping by type III restriction enzyme [74] ・Biotinylated DNA-streptavidin interaction [75] Miles Miles Shinohara Takeyasu Trimitsu 2008 5 ・Anisotropic diffusion of point defects in streptavidin 2D crystals [76] ・Identification of intrinsically disordered regions of proteins [77] ・Human chromosomes in liquid [78] ・DNA-nuclease interaction [ ・Dynamic equilibrium at the edge of bR 2D crystals [81] ・Structural change of CaM and actin polymerization on streptavidin 2D crystals [82] ・Unidirectional translocation of cellulase along cellulose fibers [83] ・Translocation of EcoRII restriction enzyme along DNA [84] ・Fabrication and imaging of hard material surface [85] ・Purple membrane in contact-mode HS-AFM [86] ・Thermal motion of π-conjugated polymer chain [87] ・Opening of 3D hollow structure of DNA Origami [88] ・Opening of 3D hollow structure of DNA Origami [89] ・ATP-induced conformational change in P2X 4 ・Walking myosin V along an actin filament [91] ・Photo-induced structural change in bR [92] ・2D crystal formation of annexin A-V and height change of p97 [93] ・Analysis of components covering magnesotome surface [94] ・Time course of cell death by antimicrobial peptide [95] ・Dissolution of extreme UV exposed resist films under developing [96] ・Dissolution of extreme UV exposed resist films under developing [97] ・Process of forming supported planar lipid bilayer [98] ・Self assembly of amyloid-like fibrils [99] ・Effect of ClpX on FtsZ polymerization ...…”

“…High-speed AFM has recently demonstrated its ability to visualize the thin and random structures of IDPs, using FACT protein as a test sample [77]. Displacement of histone H2A/H2B dimers from nucleosomes by FACT protein facilitates RNA polymerase II transcription [147] and chromatin remodeling [148].…”

High-speed atomic force microscopy and its future prospects

“…Since the oscillation amplitude of shorter cantilevers can be more precisely and sensitively detected than that of conventional long cantilevers, the vertical resolution of HS-AFM is higher than that of slow AFM, even at high bandwidth. The vertical resolution of~0.15 nm is high enough to visualize unstructured polypeptides (0.45-0.5 nm in diameter) on a solid support (Miyagi et al 2008).…”