Nanovisualization of Proteins in Action Using High-Speed AFM

The dynamics of proteins, such as their conformational change and dynamic interplay with interaction partners, is crucial to their biological functions. To reveal the dynamic behavior of proteins, single-molecule approaches are indispensable because the molecules behave in an unsynchronized manner, which makes it difficult to monitor their dynamics by ensemble average experiments. Fluorescence microscopy has been widely used to study the functional behavior of single-protein molecules (Peterman et al. 2004; Joo et al. 2008; Roy et al. 2008; Yanagida and Ishii 2008). However, it only visualizes featureless fluorescent spots, meaning that protein molecules themselves are invisible. Moreover, what we can analyze using fluorescence techniques is limited to the behavior of a selected portion where a fluorophore is attached. Therefore, inferences have to be made to bridge the gap between the observed behavior of fluorescent spots and the actual behavior of labeled protein molecules. Consequently, it takes a considerably long time to reach a persuasive conclusion on how a protein dynamically behaves to function. Atomic force microscopy (AFM) (Binnig et al. 1986) has made it possible for the first time to visualize the surface topography of samples at single-nanometer resolution even in liquid environments (Drake et al. 1989; Weisenhorn et al. 1989). In the early stage, pioneering studies explored this new capability by visualizing various biological samples under physiological conditions (Lindsay et al. 1989;Weisenhorn et al. 1989; Hansma et al. 1993; Schabert and Engel 1994; Müller et al. 1995; Shao