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

开发最高速原子力显微镜并阐明生物分子马达的纳米结构动力学

基本信息

批准号：
15101005
负责人：
ANDO Toshio
金额：
$ 58.41万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (S)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2007
项目状态：
已结题

项目摘要

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

仪器：我们开发了轻敲模式原子显微镜 (AFM) 中包含的各种设备，以获得高速旋转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谐振频率为 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超过 500 kHz 的带宽。开发了用于抑制扫描仪机械振动的主动阻尼技术，包括可以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，其增益参数可以根据悬臂振荡幅度自动改变，可以使用幅度设置非常接近悬臂自由振荡幅度的点此功能可确保从振荡悬臂尖端加载到样品上的力非常小，并且即使在样品扫描速度非常快时也可以避免尖端“跳伞”。用于悬臂激发效率漂移的补偿器允许长时间保持这种小力。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，而不会损坏脆弱的样品。正如马内所提到的，各种动态生物分子过程已成功地通过视频捕捉到。此外，我们还开发了一种用于相衬成像的快速相位检测器。该装置可以在每个振荡周期以及一个周期内的任意时刻检测悬臂振荡的相位变化。这种能力使我们能够区分能量保守和耗散的尖端-样本相互作用。因此，它可以同时对材料特性和形貌的异质性进行成像。生物成像：（1）肌球蛋白V 双头肌球蛋白V与肌动蛋白丝的核苷酸依赖性关联首先通过高速MM成像进行分析。在严格状态和ADP存在下，两个头中只有一个与F-肌动蛋白结合。从结合头的箭头结构，结合头被识别为尾部珠子。在模拟 ADP-Pi 的中等浓度 AMP-PNP 存在的情况下，两个头都与相同的肌动蛋白丝结合。因此，AMP-PNP 的结合改变了前导珠的构型，使其肌动蛋白结合位点可以面向肌动蛋白丝。在 ATP 存在的情况下，所需的肌球蛋白 V 被视频捕获；两个杠杆臂交替改变前导位置和尾随位置（即，手拉手运动）。在尾部头与肌动蛋白分离之前，前杠杆臂向前弯曲。这种弯曲导致尾部杠杆臂被向前拉，这加速了 ADP 从尾部头的解离，并促进 ATP 与尾部珠的结合，导致尾部头从肌动蛋白解离。因此，这些成像研究阐明了肌球蛋白 V 在肌动蛋白轨道上持续运动的分子机制。 (2)动力蛋白。单头动力蛋白 C 存在于 ATP 中。茎部分在两个不同的两个位置之间周期性移动，而在茎和结构蛋白 C 的主体中没有检测到明显的运动。成功地对沿着微管的酵母细胞质动力蛋白（双头）的持续成像。 (3) Chaperonin 切换 GroES 结合和未结合状态，正如 GroEL 两个环之间的负协同性（关于 ATPase 反应）所预期的那样。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存在争议。高速AFM成像立即迅速解决了这个有争议的问题。 (4)二维蛋白质晶体缺陷。对含有生物素的脂质双层上的链霉亲和素二维晶体中的移动点缺陷进行成像。其分析阐明了无缺陷蛋白质二维结晶的机制。较少的