| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1998: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1997: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
It is widely accepted that atomic force microscopy (AFM) is now a very useful tool in structural biology. However, the high-resolution AFM for biological molecules is still not so easy and not established, since proteins are too soft in water to be scanned with a tip without deformation. In this project the high-resolution AFM of two-dimensional (2D) arrays of proteins and single molecules were purposed.
Based on the previous studies, a carbonaceous tip called supertip was fabricated and used instead of commercially available silicon nitride tip, and 2D crystal specimens of globular proteins, ferritin and catalase, were prepared by means of Langmuir technique and transferred onto a surface of silicon.
A contact mode AFM using a soft cantilever (k=0.09 N/m) provided high-resolution images of 2D arrays of ferritin and catalase. Imaging in water of 2D protein arrays proved to be superior to that in air for a dried sample. This is due probably to the lack or reduction of frictional forces between tip and the water-bound protein surface.
Finally, high-resolution single molecule imaging of protein was attempted. The strategy taken for this purpose was to embed protein of interest into 2D arrays of smaller proteins: ferritin was embedded into catalase arrays and single catalase molecules were done in 2D arrays of streptavidin. With this preparation, the surface profiles of single molecules were reduced and they were well immobilized. The AFM images were of convolution between the globular proteins of about 10 nm and the tip with the end radius of 3 nm.
For higher-resolution imaging, which manifests subunit structure of single molecules, technical developments in tip fabrication and more sophisticated control of the tip during scanning is required. The immobilization method developed here is though to be useful for this purpose in future.
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