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

• Published in: Chemphyschem 2008-09, Vol.9 (13), p.1859-1866
• Main Authors: Miyagi, Atsushi, Tsunaka , Yasuo, Uchihashi , Takayuki, Mayanagi, Kouta, Hirose, Susumu, Morikawa , Kosuke, Ando , Toshio
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; atomic force microscopy; Biological and medical sciences; biophysics; Fundamental and applied biological sciences. Psychology; Gene Deletion; Medical sciences; Microscopy, Atomic Force - methods; Microscopy, Electron, Transmission; Mutation - genetics; protein modifications; protein structures; Proteins; Proteins - genetics; Proteins - ultrastructure; Time Factors; Toxicology
• ISSN: 1439-4235; 1439-7641
• DOI: 10.1002/cphc.200800210

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. High‐speed AFM imaging of the heterodimeric FACT (facilitates chromatin transcription) protein reveals two tail‐like structures protruding from its main body (see schematic picture). These very thin and undulating tail segments are identified as the intrinsically disordered regions of FACT predicted from the amino acid sequences, the mechanical properties of which, estimated from the AFM images, suggest that they have more relaxed structures than random coils.