| Project/Area Number |
14380284
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | University of Tsukuba |
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Principal Investigator |
TANAKA Toshiyuki University of Tsukuba, Graduate School of Life and Environmental Sciences, Professor, 大学院生命環境科学研究科, 教授 (10217052)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥15,000,000 (Direct Cost: ¥15,000,000)
Fiscal Year 2004: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2003: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2002: ¥9,300,000 (Direct Cost: ¥9,300,000)
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| Keywords | chromoprotein / antibiotics / molecular recognition / site-directed mutagenesis / NMR / calorimetry |
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| Research Abstract |
C-1027 is one of the most potent antitumor antibiotic chromoproteins, and is a 1:1 complex of an enediyne chromophore having DNA-cleaving ability and a carrier apoprotein. The purpose of this project is to understand the mechanisms for the recognition and the release of chromophore by the apoprotein.
We prepared the ^<13>C/^<15>N-labelled apoprotein and determined the three-dimensional solution structure of C-1027 apoprotein and its complex with the aromatized chromophore. Based on these high-resolution structures, we were able to elucidate the possible binding interactions between the chromophore and the apoprotein. To confirm the possible electrostatic interactions, we performed the binding experiments by calorimetry using the aromatized chromophore and a series of mutant apoproteins, of which an acidic, basic, or hydrophilic amino acid residue is replaced with the different type of amino acids. From the binding experiments, we concluded that the most important electrostatic interaction is the salt bridge between the Asp101 carboxylate and the 18-N^+H_3 of the chromophore and that the interaction is modulated by the nearby imidazole ring of His104. We also analyzed the backbone dynamics of the apoprotein in both free and bound states and were able to specify the amino acid residues that play an important role in releasing the chromophore. Finally, we succeeded in creating the mutant proteins that could bind two molecules of ethidium bromide, which the natural apoprotein never binds.
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