SEQUENTIAL H-1-NMR ASSIGNMENT OF THE COMPLEX OF APONEOCARZINOSTATIN WITH ETHIDIUM-BROMIDE AND INVESTIGATION OF PROTEIN DRUG-INTERACTIONS INTHE CHROMOPHORE BINDING-SITE

Two-dimensional H-1 NMR spectroscopy has been used to investigate the binding site, binding interactions, and the conformation of a 1:1 comp lex of aponeocarzinostatin (apo-NCS) with ethidium bromide in solution . The protein component in the complex has been sequence-specifically assigned using information derived from coherence transfer and two-dim ensional homonuclear H-1 NOESY experiments. The conformation of the pr otein in the complex has been found to be similar tb the free form of the apoprotein, and intermolecular NOEs between the residues of the pr otein to protons on the ethidium bromide suggest that the ethidium bro mide is bound to the protein in the same cleft in which the neocarzino statin chromophore binds. Protons on ethidium show NOE interactions to the following protein residues: Trp-39, Leu-45, Cys-47, and Gln-94 wh ich interact With the phenanthridine ring system of ethidium, Gly-102 and Asn-103 which interact with the alkyl chain of ethidium, and Phe-5 2 which interacts with the phenyl ring of ethidium. The orientation of ethidium in the cleft of apo-NCS is compared and contrasted to orient ation of the chromophore as determined by high-resolution NMR and X-ra y diffraction studies.