Effect of Variants at the 78th Residue on Neocarzinostatin Chromophore Release

Abstract

Neocarzinostatin is a potent antitumor antibiotics chromoprotein complex. It consists of a labile enediyne chromophore and a protective carrier protein. Release of the bioactive chromophore is the initial key step in its cytotoxic mode of action. We showed that F78 of the carrier protein plays an important role in gating the release. To further study the structural role of 78th side chain in the release mechanism, we made variants at the 78th residue using a PCR-based in vitro mutagenesis method. The identity and purity of the expressed protein variants were confirmed by SDS-PAGE, UV, HPLC, and MS spectroscopy. Oxygenation was applied to promote formation of disulfide bonds when necessary, and the integrity of disulfide linkages was verified by a designed iodoacetamide-based test. Thermal denaturation study and CD spectroscopic analysis showed that stability and backbone conformation of these variants were conserved as compared to the wild-type protein. Two-dimensional 15N-1H HSQC NMR study suggested that the majority of residues around the binding cleft were not disturbed. Reconstitution of the enediyne chromophore into these protein variants showed efficient binding and the binding structure appeared to be similar to that of the native neocarzinostatin. Once the backbone structural effect was excluded, side chain structural effect on kinetic release was studied by monitoring fluorescence changes on samples containing glutathione and the reconstituted variants. The results showed that, except aromatic side chains, the basic variants increased the release rate more significantly than the acidic ones. Depended upon the length, steric hindrance, or hydrophobicity, the variants with aliphatic side chain also increased the release rate at different extent.