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  • Title: Actinomycin D-deoxynucleotide complexes as models for the actinomycin D-DNA complex. The use of nuclear magnetic resonance to determine the stoichiometry and the geometry of the complexes.
    Author: Krugh TR, Chen YC.
    Journal: Biochemistry; 1975 Nov 04; 14(22):4912-22. PubMed ID: 1182128.
    Abstract:
    The use of proton and carbon-13 magnetic resonance spectroscopy for the determination of the geometry and the stoichiometry of the actinomycin D-deoxyguanosine 5'-monophosphate complex is outlined. The dimerization of actinomycin D has been reexamined by recording the proton magnetic resonance spectrum of actinomycin D to much lower concentrations through the use of Fourier transform nuclear magnetic resonance techniques. The effect of the actinomycin D dimerization on the observed chemical shifts that results from the additon of nucleotides to an actinomycin D solution is directly demonstrated by comparing the actinomycin D-nucleotide titrations at both low (approximately 0.3 mM) and high (approximately 12 mM) concentrations of actinomycin D. In the presence of excess nucleotide the chemical shifts of the actinomycin D groups were essentially the same for both the low and high concentration titrations. The complexes of actinomycin D with pdG-dC, dG-dC, deoxyguanosine 3'-monophosphate, G-C, C-G, dIMP(5'), 2, 6-diaminopurine deoxyribose, and other nucleotides were also investigated by proton magnetic resonance and visible spectral titrations. These data were interpreted in terms of the molecular geometry of the complexes and in terms of the effect of the structure of the nucleotide base on the relative binding affinity of the nucleotides for the two nucleotide binding sites of actinomycin D. The carbon-13 chemical shifts of dGMP(5') were measured as a function of concentration over the concentration range of 0.5-0.025 M. The infinite dilution carbon-13 chemical shifts were graphically estimated from the dilution curves. These values were used to calculate the changes in the chemical shifts of the dGMP carbons that result from the formation of an actinomycin D-(dGMP)2 complex. It was not possible to interpret these carbon-13 chemical shift changes in terms of only ring current effects, which thus rules out the use of carbon-13 spectroscopy in the determination of the geometries of the actinomycin D complexes with the mono- and dinucleotides. The induced chemical shifts in the proton spectra may be used in the determination of the geometries of the complexes. A consideration of these data for the above nucleotide series shows that the predominant complex formed is one in which the guanine rings in the two nucleotide binding sites of actinomycin D are oriented in a manner very similar to that observed in the cocrystalline complex of actinomycin D with deoxyguanosine.
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