|Budget Amount *help
¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 2005 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 2004 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 2003 : ¥1,500,000 (Direct Cost : ¥1,500,000)
The structure of ATP receptor/channel (P2X receptor) was observed using atomic force microscopy. When receptor proteins prepared from insect-derived Sf9 cell expression system were observed in air, flat island-like structures of about 4 nm in height were seen. For observation in water, images of individual receptor proteins were obtained, and their height was found to be about 4 nm. When ATP was added to the water, the ratio of receptor proteins forming aggregations was increased, suggesting that the structure of the proteins are changed in the presence of ATP. When observation was made in the water with a higher ionic strength, two-dimensional crystal-like images where the proteins were densely packed in the presence of ATP were obtained. Such densely packed proteins can provide higher resolution because of reduced movement of probes along the z-axis. Actually, the surface of the receptor proteins could be analyzed with nanometer resolution. The analysis provided the following information : 1) the proteins were composed of a unit structure with 10 nm in diameter, 2) a pore structure is found in the middle of the unit structure, and 3) each unit consists of several (maybe three) subunits. Such densely packed images were not obtained in the absence of ATP, suggesting that the structure of the receptor proteins is not uniform when they are not occupied by ATP. When suramin, an antagonistat ATPreceptors, was applied in the presence of ATP, densely packed images were obtained, but the unit structure in the surface was not obvious. This result suggests that : 1) suramin does not simply replace with ATP, and 2) the structure of suramin-bound receptor proteins is different from that of ATP-bound ones. In summary, these findings indicate that atomic force microscopy is a powerful tool for "nano" level analysis of interactions between receptor proteins and ligands.