High-speed atomic force microscopy (HS-AFM) is a unique tool that enables imaging of protein molecules during their functional activity at sub-100 ms temporal and submolecular spatial resolution. HS-AFM is suited for the study of highly dynamic proteins, including myosin motors. HS-AFM images of myosin V walking on actin filaments provide irrefutable evidence for the swinging lever arm motion propelling the molecule forward. Moreover, molecular behaviors that have not been noticed before are also displayed on the AFM movies. This chapter describes the principle, underlying techniques and performance of HS-AFM, filmed images of myosin V, and mechanistic insights into myosin motility provided from the filmed images.
Keywords: Chemo-mechanical coupling; High-speed atomic force microscopy; Molecular motor; Myosin ATPase; Single-molecule measurement.