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Visualization of Hydrophobic-, Hydrophilic-, and Charged Areas of Protein by Photochromic Cantilevers.

通过光致变色悬臂可视化蛋白质的疏水、亲水和带电区域。

基本信息

批准号：
10640384
负责人：
ANDO Toshio
金额：
$ 2.11万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Atomic force microscope (AFM) was first invented as an imaging tool for nanometer worlds. However, it can be used also as a high-sensitive force sensor, which allows us to obtain a 2D map of forces exerted between a cantilever probe and sample surface. Adhesive, repulsive, rupture, or non-contact forces between an AFM probe and sample can tell us physicochemical properties of the sample such as hydrophobicity, hydrophilicity and electric charges, depending on the surface property of the probe used. To specify the physicochemical property of sample clearly it is best to scan the sample with probes having different properties. Yet, it is quite difficult to scan the same area of sample after changing cantilevers. This has been a main reason why AFM has not been successfully used for mapping property of protein surface. To detour this problem we developed an alternative method. Instead of changing cantilevers, we change the surface property of a photochromic probe by irradiating near-UV li … More ght. We synthesized a photochromic dye, vinyl malachite green (VMG) whose structure can change from hydrophobic to charged form on irradiation. This dye was attached to the tip of cantilever probes covalently. We also invented a scanning method to map rupture forces within a time shorter than an ordinary force-curve method. Although it was shorter, it still took 30 min, resulting in suffering from disturbance by mechanical and electrical drifts of the AFM.After confirming the usefulness of the photochromic probe in identifying physicochemical property of sample, we invented an additional scanning method to obtain non-contact force map and topography simultaneously. This method shortened the scan time to 5 min (although it is not short enough yet). The positive charges on a basic protein, lysozyme, was successfully visualized by this method. Parallel to these studies, we have developed a high-speed AFM.It can ultimately solve the problem that the observation of force map requires much longer time than topography. The frame rate for topography observation reached 2.5 frames/sec, about 250-times faster than ordinary AFM apparatus. Although we have not tried to use this new AFM for force mapping, its speed promises a few seconds of force mapping, and therefore in the near future we may be able to obtain much better resolution of force map on protein surfaces. Less

原子力显微镜（AFM）最初是作为纳米世界的成像工具而发明的。然而，它也可以用作高灵敏度的力传感器，这使我们能够获得悬臂探针和样品表面之间施加的力的2D图。AFM探针和样品之间的粘附力、排斥力、破裂力或非接触力可以告诉我们样品的物理化学性质，如疏水性、亲水性和电荷，这取决于所使用的探针的表面性质。为了明确样品的物理化学性质，最好用具有不同性质的探针扫描样品。然而，在更换样品杆后，很难扫描样品的相同区域。这是AFM未能成功应用于蛋白质表面性质的主要原因。为了避开这个问题，我们开发了另一种方法。我们用近紫外光照射改变光致变色探针的表面性质， ...更多信息好的。合成了一种光致变色染料乙烯基孔雀石绿色（VMG），其结构在光照下可由疏水性转变为荷电性。这种染料共价连接到悬臂梁探针的尖端。我们还发明了一种扫描方法，可以在比普通力曲线方法更短的时间内绘制破裂力。虽然时间较短，但仍然需要30 min，导致AFM受到机械和电漂移的干扰。在确认了光致变色探针在识别样品物理化学性质方面的有用性后，我们发明了一种额外的扫描方法，以同时获得非接触力图和形貌。这种方法将扫描时间缩短到5分钟（尽管还不够短）。用这种方法成功地显示了碱性蛋白质溶菌酶上的正电荷。与此同时，我们研制了一台高速原子力显微镜，解决了力图观测时间远长于形貌观测时间的问题。用于形貌观察的帧速率达到2.5帧/秒，比普通AFM设备快约250倍。虽然我们还没有尝试使用这种新的AFM进行力映射，但它的速度保证了几秒钟的力映射，因此在不久的将来，我们可能能够在蛋白质表面获得更好的力映射分辨率。少