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

仪器：我们开发了包含在轻拍模式原子闹剧显微镜(AFM)中的各种设备，以获得高速漫游能力以及对样品的低侵入性。与奥林巴斯合作开发的小悬臂在水中的共振频率为1.2 MHz，弹簧常数为02 N/m。z扫描仪的带宽达到了前所未有的带宽，超过500 kHz。用于抑制扫描仪机械振动的主动减振技术，包括一种能自动产生给定传递函数的逆传递函数的电路。用于检测悬臂挠度的位置传感器的带宽已达到20 MHz。动态PID控制器的增益参数可根据悬臂振荡幅度自动改变，使幅度设定值非常接近悬臂的自由振荡幅度。这种能力确保了非常小的力…更多的载荷从摆动的悬臂尖端加载到样品上，即使在以非常快的速度扫描样品时，也可以避免尖端的“跳伞”。悬臂激发效率漂移的补偿器使这种小作用力可以保持很长时间。配备这些设备的高速AFM可以以30-60 mA/帧的成像速率捕获图像，而不会损坏易碎的样品。“正如下面提到的，各种动态的生物分子过程已经成功地被视频捕捉到。此外，我们还研制了一种用于相衬成像的快速鉴相器。该装置可以检测悬臂梁振动在每个振荡周期和任意时刻的相位变化。这种能力使我们能够区分能量守恒和耗散的针尖-样品相互作用。生物成像：(1)肌球蛋白V双头肌球蛋白V与肌动蛋白细丝的核苷酸依赖的结合首先在僵硬状态下用高速MM成像分析，在ADP存在的情况下，两个头中只有一个与F-肌动蛋白结合。从被捆绑的头部的箭头结构中，被捆绑的头部被识别为拖珠。在中等浓度的模拟ADP-PI的AMP-PNP存在下，两个头结合到相同的肌动蛋白细丝上。因此，AMP-PNP的结合改变了前导微珠的构型，使其肌动蛋白结合部位可以面对肌动蛋白细丝。在三磷酸腺苷存在的情况下，视频捕捉到了想要的肌球蛋白V；两个杠杆臂交替地改变领先和落后的位置(即，双手上手运动)。在拖尾与肌动蛋白分离之前，前导杠杆臂向前弯曲，导致拖尾杠杆臂被向前拉，这加速了ADP从拖尾头部解离，并促进了ATP与拖尾微珠的结合，导致拖尾头部与肌动蛋白解离。因此，这些成像研究阐明了肌球蛋白V在肌动蛋白轨迹上进行移动的分子机制。(2)Dynein。单头动力蛋白C在三磷酸腺苷存在下。茎部分在两个不同的位置之间周期性地移动，而茎和粘连蛋白C的主体没有明显的移动。成功地成像了酵母胞质动力蛋白(双头)沿微管的过程。(3)伴侣素在GroEL的两个环之间的负协同性(关于ATPase反应)中切换Groes结合和非结合状态，这是预期的。然而，在转换之前，出现了Groes-GroEL-Groes复合体。这种复杂的阵型长期以来一直备受争议。高速原子力显微镜成像技术迅速解决了这个有争议的问题。(4)2D蛋白晶体缺陷。对链霉亲和素2D晶体在生物素脂类双层膜上的移动点缺陷进行了成像。它的分析阐明了无缺陷蛋白质2D结晶的机制。较少

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