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Abstract
Neocarzinostatin is a potent enediyne antitumor antibiotic complex in which a chromophore is noncovalently bound to a carrier protein. The protein regulates availability of the drug by proper release of the biologically active chromophore. To understand the physiological mechanism of the drug delivery system, we have examined the trifluoroethanol (TFE)-induced conformational changes of the protein with special emphasis on their relation to the release of the chromophore from holoneocarzinostatin. The effect of the alpha helix-inducing agent, TFE, on all the beta-sheet neocarzinostatin proteins was studied by circular dichroism, fluorescence, and (1)H NMR studies. By using binding of anilinonaphthalene sulfonic acid as a probe, we observed that the protein exists in a stable, partially structured intermediate state around 45-50% TFE, which is consistent with the results from tryptophan fluorescence and circular dichroism studies. The native state is stable until 20% TFE and is half-converted into the intermediate state at 30% TFE, which starts to collapse beyond 50%. High pressure liquid chromatographic analysis of the release of the chromophore caused by TFE treatment at 0 degrees C suggests that the release process, which occurs below 20% TFE, does not result from an observable conformational change in the protein. Kinetic measurements of the release of chromophore at 25 degrees C reveal that TFE does stimulate the rate of release, which increases sharply at 15% and reaches a maximum at 20% TFE, although no major secondary or tertiary structural change of the carrier protein is observed under these same conditions. Our data suggest that chromophore release results from a fluctuation of the protein structure that is stimulated by TFE. Complete release of the chromophore occurs at TFE concentrations where no overall observable unfolding of the apoprotein is seen. Thus, the results suggest that denaturation of the protein by TFE is not a necessary step for release of the tightly bound chromophore.
Highlights
The biological function of the carrier apoprotein of the enediyne chromoprotein, which is a recently discovered natu
Circular Dichroism—CD spectra were recorded at both nearand far-UV to monitor the conformational changes induced by titration of TFE
Since direct binding of NCS protein to DNA has not been observed [13, 14], release of the active chromophore has to be the first step in its pharmacological action of cleaving DNA
Summary
The biological function of the carrier apoprotein of the enediyne chromoprotein, which is a recently discovered natu-. One important pharmacological function is that the protein regulates availability of the drug by proper release of its biologically potent chromophore. To understand the physiological mechanism of the natural drug delivery system, it is interesting and essential to study how the protein releases the tightly bound chromophore. Neocarzinostatin (NCS) is the first enediyne chromoprotein; it consists of a labile chromophore (NCS-Chr) (Mr ϭ 659, Fig. 1) [10, 11] that is tightly and noncovalently bound to an apoprotein (apoNCS) (113 amino acids) [12]. It has been reported earlier that DNA cleavage induced by native NCS can be greatly stimulated by organic solvents and denaturants in vitro (14 – 16) This effect might well result from a denaturation process that releases the active chromophore. The amino acid residues that form the hydrophobic binding pocket were assigned later by three-dimensional structural studies [23,24,25,26,27,28,29,30,31,32,33,34,35,36,37, 39]
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