Development of a highest-speed atomic force microscope and elucidation of the nano-structural dynamics of biological Molecular motors

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Instrumentation: We developed various devices contained in the tapping mode atomic farce microscope (AFM) to attain a high-speed roan capability as well as low invasiveness to the sample. The small cantilevers developed collaborating with Olympus have a resonant frequency of 1.2 MHz in water and a spring constant of 02 N/m. The bandwidth of the z-scanner has reached an unprecedented bandwidth beyond 500 kHz. Active damping techniques for suppressing the scanner's mechanical vibrations, including an electric circuit which could automatically produce an inverse transfer function of a given transfer function, were developed L The bandwidth of a position sensor for detecting the cantilever deflection has reached 20 MHz The dynamic PID controller, whose gain parameters can be automatically changed depending on the cantilever oscillation amplitude, enables the use of an amplitude set point very close to the free oscillation amplitude of a cantilever This capability ensures a very small force … More loaded onto the sample from the oscillating cantilever tip and can avoid 'parachuting' of the tip even when the sample is scanned very fast A compensator for drift in the cantilever excitation efficiency allows this small force to be maintained for a long time. The high-speed AFM equipped with these devices can capture an image at an imaging rate of 30-60 ma/frame without damaging the fragile samples. 'Mane as mentioned below various dynamic biomolecular processes has successfully been captured on video. In addition, we developed a fast phase detector for phase contrast imaging. This device can detect the phase change in the cantilever oscillation at every oscillation cycle and at an arbitrary timing with in a cycle. This capability allows us to distinguish the energy conservative and dissipative tip-sample interactions. Therefore, it can simultaneously image heterogeneity of material properties and the topography.Bioimaging: (1) Myosin V The nucleotide-dependent association of double-headed myosin V to actin filaments was first anamyzed by high-speed MM imaging In the rigor state and in the presence of ADP only one of the two heads was bound to F-actin. From the arrow-head structure of the bound head, the bound head was identified to the trailing bead. In the presence of a medium concentration of AMP-PNP which mimics ADP-Pi, the both heads were bound to the same actin filament. Therefore, the binding of AMP-PNP changes the leading bead configuration so that its actin binding site can face an actin filament. In the presence of ATP, the wanting myosin V was captured on video; the two lever arms change the leading and trailing positions alternately (ie., hand-overhand movement). Before the trailing head detached from actin, the leading lever arm bent frontward This bending results in the trailing lever arm being pulled frontward, which accelerates the ADP dissociation from the trailing head and facilitates ATP binding to the trailing bead leading to the dissociation of the trailing head from the actin. Thus, these imaging studies elucidated the molecular mechanism for the processive movement of myosin V on actin track. (2) Dynein. Single-headed dynein C was in the presence of ATP. The stem moiety moved periodically between two distinct two positions, while no apparent movement was detected in the stalk and the main body of denein C. The processive of yeast cytoplasmic dynein (two-headed) along microtubules was successfully imaged. (3) Chaperonin switching the GroES bound and unbound states, as expected from the negative cooperativity (regarding the ATPase reaction) between the two rings of GroEL. However, a GroES-GroEL-GroES complex appeared before the switching. This complex formation had been controversial for a long time. High-speed AFM imaging quickly solved this controversial issue instantly. (4)Defect in 2D protein crystal. Moving point defects in streptavidin 2D crystal on biotin containing lipid bilayers was imaged. Its analysis elucidated the mechanism of defect-free protein 2D crystallization. Less