¹H NMR Studies of the Binding of Bacteriophage‐M13‐Encoded Gene‐5 Protein to Oligo(deoxyadeny1ic acid)s of Varying Length

Abstract

The binding of gene-5 protein to oligo(deoxyadeny1ic acid)s varying in length from 2 to 16 nucleotides has been studied by titrating the protein with the oligonucleotides and recording the 'H 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 Overhauser 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) Biochemisrry, 20, 4419-44281. The 'H NMR spectra of the complexes of gene-5 protein with (dA)n, (dA)Il and (dA)16 appear to be identical except for differences in linewidth. The 'H NMR spectra of the complexes with the smaller oligonucleotides (dA)2, (dA)3 and (dA), 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.Gene-5 protein is a DNA-helix-destabilizing protein encoded by the Escherichia coli phages M13 f l and fd. Since its first isolation by Alberts et al. [I] and Oey and Knippers [2] a considerable amount of information has become available.Physico-chemical studies have revealed that the protein exists in solution mainly as a dimer [2-41 and binds in a cooperative manner to single-stranded DNA thereby covering about four nucleotides per protein monomer [3,5].Crystallographic studies [6,7] have disclosed that the peptide backbone consists entirely of antiparallel 8-structure. The DNA binding sites are observed as 3-nm-long grooves, having opposite polarity in the two halves of the dimer. The elucidation of the crystal structure of protein-oligonucleotide complexes is in progress [8]. In the electron microscope the gene-5-protein . DNA complex is observed as a helical rod with a width of 10 nm and a mean helical pitch of 9.1 nm [9]. Circular dichroism [5] and NMR studies [lo-171 have provided evidence for the involvement of tyrosines and phenylalanines in the DNA binding. Recently we arrived at a complete assignment of the aromatic 'H NMR spectrum of gene-5 protein by performing selective decoupling and nuclear Overhauser enhancements at 500 MHz [16]. The assignments are in agreement with earlier assignments based on photo-chemically induced dynamic nuclear polarization [I 1,131 and selective deuteration studies [14]. In addition an interpretation could be given of the 500-MHz 'H NMR spectrum of the gene-5-protein . (dA)e complex, which rests in part on the titration studies presented in this paper. It was concluded from the 500-MHz s...

Fluorescence studies of the binding of bacteriophage M13 gene V mutant proteins to polynucleotides

“…The aromatic residues Y26, Y34, and Y41 of one monomer and F73 of the opposite monomer in the dimer are involved in the binding by stacking with the bases of the nucleic acid. The residues R16, R21, R80 and K46 were believed to be involved in the binding by ionic interactions with the phosphate moieties of the nucleic acid.In solution the structure of gVp and of its complex with oligonucleotides has been studied extensively by means of 'H-NMR techniques [13,. These experiments have shown that the structure of these complexes is different from the structure proposed by Brayer and McPherson.…”

“…In solution the structure of gVp and of its complex with oligonucleotides has been studied extensively by means of 'H-NMR techniques [13,. These experiments have shown that the structure of these complexes is different from the structure proposed by Brayer and McPherson.…”

“…'H-NMR studies [13] have revealed that, on binding to oligonucleotides, the number of nucleotides/protein monomer is 3.…”

“…In the life cycle of the phage, it fulfils at least two essential functions. First, gVp switches, late in infection when its concentration has reached a certain critical threshold level, the replication of the phage genome from the replicative formation to the progeny ssDNA formation by binding in a cooperative way to the ssDNA of the phage (for a recent review, see [l] 'H-NMR studies [13] have revealed that, on binding to oligonucleotides, the number of nucleotides/protein monomer is 3. The structure of gVp has been determined crystallographically by Brayer and McPherson [14].…”

Sequence‐specific 1H‐NMR assignment and secondary structure of the Tyr41 → His mutant of the single‐stranded DNA binding protein, gene V protein, encoded by the filamentous bacteriophage M13

“…The assignments obtained for GVP Y41H can thus be used to identify amino acid proton resonances of the wild-type molecule to evaluate for example the effects of oligonucleotide binding [26]. Small but significant differences in a-resonance chemical shifts ( > 0.03 and < 0.12 ppm) between the two molecules were observed for residues 39,40,42 and 43.…”