A 500-MHz proton nuclear magnetic resonance study of the structure and structural alterations of gene-5 protein-oligo(deoxyadenylic acid) complexes.

The complex of the gene-5 protein of bacteriophage M13 with octadeoxyadenylic acid [d(A)8] has been shown earlier to differ in various respects from the complex with polynucleotides [Alma, N. C. M., Harmsen, B. J. M., van Boom, J. H., van der Marel, G., & Hilbers, C. W. (1982) Eur. J. Biochem. 122, 319-326]. In this paper the gene-5 protein-d(A)8 complex is compared with the complex formation between the gene-5 protein and a mixture of longer oligonucleotides, i.e., d(A)25-30. Nuclear Overhauser experiments have been performed on both systems to obtain structural information regarding the oligonucleotide protein interactions. In the experiments also oligonucleotides deuterated at the adenyl C8 positions have been used in order to distinguish between the adenyl H2 and H8 resonances. Combination of the experiments shows that the nucleotides in the complexes are situated in such a way that the adenyl H8 and the sugar H1' protons are near the protein surface while the adenyl H2 protons are relatively far removed from all other protons, indicating that this side of the base is pointing away from the protein surface. It is concluded that structurally different complexes can be obtained for the d(A)25-30 system. The complex with d(A)25-30 undergoes a structural transition when going from excess oligonucleotide to excess gene-5 protein. This transition is identified with the transition between the "oligonucleotide" and the "polynucleotide" binding mode. The information derived from the present NMR experiments combined with known data from X-ray diffraction and electron microscopic studies is used to propose a model for a possible orientation of the adenyl bases in the complex.

1H NMR studies of the binding of bacteriophage-M13-encoded gene-5 protein to oligo(deoxyadenylic acid)s of varying length.

The binding of gene-5 protein to oligo(deoxyadenylic acid)s varying in length from 2 to 16 nucleotides has been studied by titrating the protein with the oligonucleotides and recording the 1H NMR spectra at 360 MHz. To obtain information about the mode of binding of the protein the aromatic parts of the spectra have been analysed by performing spectral simulations, starting from the assignments obtained from nuclear Overhausfer enhancements at 500 MHz [Alma, N. C. M., Harmsen, B. J. M., Hull, W. E., Van der Marel, G., Van Boom, J.H., and Hilbers, C. W. (1981) Biochemistry, 20, 4419-4428]. The 1H NMR spectra of the complexes of gene-5 protein with (dA)8, (dA)12 and (dA)16 appear to be identical except for differences in linewidth. The 1H NMR spectra of the complexes with the smaller oligonucleotides (dA)2, (dA)3 and (dA)4 differ from each other and from the spectra obtained from the complexes with longer oligonucleotides. However, binding of all oligonucleotides basically influences the same aromatic residues, namely two tyrosines and one phenylalanine. In the protein-oligonucleotide complexes, one protein monomer covers three nucleotide residues, in contrast to the stoichiometry of 1:4 found for protein-polynucleotide complexes. It was found that the binding to oligonucleotides is cooperative and ionic-strength-dependent but far less so than found for the binding to polynucleotides.

Double-resonance experiments at 500 MHz on gene-5 protein and its complex with octadeoxyriboadenylic acid.

In this paper, a detailed description is presented of the aromatic part of the 500-MHZ 1H nuclear magnetic resonance (NMR) spectrum of the helix-destabilizing gene-5 protein (GVP) encoded by the coliphage M13. As a result of the resolution obtained at 500 MHZ, it was possible to perform selective decoupling and time-resolved selective Overhauser experiments. The magnitudes of the observed Overhauser effects compare favorably with magnitudes expected on the basis of theoretical calculations. These experiments in conjunction with selective decoupling experiments allowed a detailed interpretation of the aromatic part of the protein spectrum. The spectrum of the aromatic part of the GVP-d(A)8 complex could be interpreted in a similar fashion. The ring protons of one phenylalanyl residue and of two tyrosyl residues show rather large shifts upon complex formation. This indicates that these residues are involved in the interaction with the DNA molecule in accordance with earlier observations. Direct evidence for the proximity of these aromatic rings and the DNA fragment in the complex was obtained by additional Overhauser experiments. It turns out that the H3',H4', and/or the H5' sugar protons of the oligonucleotide are situated near the ring protons of (most likely) two or all three of the aromatic residues of which the resonances undergo large shifts upon complex formation.