The goal of this proposal is to use high resolution two dimensional nmr methods together with distance geometry techniques to determine the solution structure of the protein anticancer drug Neocarzinostatin (NCS). NCS is a relatively large (MW=11500), acidic protein isolated from Streptomyces carzinostaticus culture media. NCS is the best studied of the Streptomyces anticancer agents partly because of its low toxicity and broad in vivo activity against human leukemia, bladder cancer, liver metastasis, and sarcoma. The active anticancer agent in NCS is a nonprotein chromophore of formula C35H33NO12. Although NCS has been studied by x-ray diffraction methods, the structures of the holoprotein, the bound chromophore, and portions of the apoprotein structure remain unclear. Using a combination of phase sensitive COSY, NOESY, RELAY, multiple quantum nmr, and isotropic mixing, the 113 spin systems of apoNCS and holoNCS will be sequentially assigned. NOE's will be used to provide distance constants for a distance geometry structural refinement. The ultimate objective of this program is to elucidate how this system specifically binds and stabilizes the chromophore, and to investigate the mechanism of action of this drug at the molecular level.
|Mohanty, S; Sieker, L C; Drobny, G P (1994) Sequential 1H NMR assignment of the complex of aponeocarzinostatin with ethidium bromide and investigation of protein-drug interactions in the chromophore binding site. Biochemistry 33:10579-90|
|Remerowski, M L; Glaser, S J; Sieker, L C et al. (1990) Sequential 1H NMR assignments and secondary structure of aponeocarzinostatin in solution. Biochemistry 29:8401-9|