| Summary: | 碩士 === 國立中興大學 === 化學系 === 93 === Neocarzinostatin, an antitumor antibiotic from Streptomyces carzinostaticus, is composed of a labile enediyne chromophore and an all-β protein with 113 amino acid residues. The biological activity in damaging DNA is a sole property of the chromophore. To perform its function, the chromophore needs to be released from the protein. The crystal structure of the chromoprotein complex shows that the pi-face of the enediyne ring in the chromophore interacts closely with the Phe78 benzene ring side chain in the protein. The reported NMR studies show that the side chain of Phe78 has multiple conformations in solution. Our accumulated data suggest that Phe78 may have an important role in controlling the release of the chromophore. The present investigation aims at elucidating how Phe78 is involved in the chromophore release. Here we use apoNCS variants at the 78th amino acid residue with alternative side chains to serve such a purpose. In this study, we choose 5 different variants with 78th side chain that is absent (G), positively charged (K), negatively charged (E), neutral but highly polared (Q), or similarly structured (Y).
In the present study, a PCR based in vitro mutagenesis method is used to construct apoNCS variants at Phe78 position. The mutant apoNCS genes are sequenced to confirm the intended mutation at the desired position. The recombinant wild-type apoNCS and its variants are purified from IPTG-induced expression of respective proteins from in E. coli The identity and purity of apoNCS variants are analyzed by SDS-PAGE, HPLC, mass spectroscopy, and disulfide linkage examination. The fluorescence-based kinetic release experiments with reconstituted holo-proteins are performed for each purified mutants as well as the recombinant wild-type protein. Measurements of initial rate reveal that some mutants, in particular F78K, can have several fold faster rate to release its chromophore than WT can. On the other hand, the circular dichroism studies show that all these mutants have similar secondary as well as tertiary structure to that of WT protein. Tm measurements suggest that thermal stability of these mutants are similar. The NMR 2D 15N-H1 HSQC experiments further confirm the close structural similarity between mutants and WT. Our results suggest that the faster chromophore release rate could be attributed to varied chemical nature of the side chain being replaced at Phe78 position, but not from significant backbone structural changes. Further NMR studies may throw in more light on the mechanism of the chromophore release.
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