Proteins are dynamic in nature, work at the single-molecule level, and facilitate several biological functions. The structure of a protein is closely associated with its function; thus, a large number of structural analyses of proteins were performed using techniques such as X-ray crystallography and NMR. Although these methods provide structural information, they often fail because of difficulties in crystallizing the proteins that are complexed with other biomolecules. Moreover, these techniques do not allow the observation of structural changes in the active form of the molecule. Single-molecule fluorescence techniques have been used for the direct observation of protein functions; however, they only reveal the dynamics of individual fluorescent spots, rather than the structural changes that occur over the entire protein. The recent development of high-speed atomic force microscopy (HS-AFM) overcame this problem and allowed the observation of the structural dynamics of proteins and other biomacromolecules directly and in real time. In this chapter, we describe the HS-AFM analysis of the dynamic molecular processes in photoactivated bacteriorhodopsin, membrane-mediated protein-protein interactions, ATP-induced conformational changes in purinergic receptors, the two-dimensional crystal structure of streptavidin, the nature of FtsZ polymers, the role of ClpX in the regulation of FtsZ polymer dynamics, the function of restriction enzymes, the action of motor proteins, the movement of TrCel7A on crystalline cellulose substrates, and the antimicrobial peptide activity on individual bacterial cells.
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