The fluorescence quenching of the bacteriophage M13 encoded gene-5 protein was used to study its binding characteristics to different polynucleotides. Experiments were performed at different salt concentrations and in some instances at different temperatures. The affinity of the protein depends on the base and sugar composition of the polynucleotides involved and may differ appreciably, i.e. by orders of magnitude. The salt dependence of binding is within experimental accuracy equal for all single stranded polynucleotides. A method is presented to estimate values of the cooperativity constant from salt titration curves. These values are systematically higher than those obtained from titration experiments in which protein is added to a polynucleotide solution. A comparison is made between the binding constants of the gene-5 protein and the gene-32 protein encoded by the T4 phage. Possible implications of the binding characteristics of the gene-5 protein for an understanding of its role in vivo are discussed.

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.

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...

This investigation describes how the binding characteristics of the single-stranded DNA-binding protein encoded by gene V of bacteriophage M13, are affected by single-site amino acid substitutions.The series of mutant proteins tested includes mutations in the purported monomer-monomer interaction region as well as mutations in the DNA-binding domain at positions which are thought to be functionally involved in monomer-monomer interaction or single-stranded DNA binding. The characteristics of the binding of the mutant proteins to the homopolynucleotides poly(dA), poly(dU) and poly(dT), were studied by means of fluorescence-titration experiments. The binding stoichiometry and fluorescence quenching of the mutant proteins are equal to, or lower than, the wild-type gene V protein values. In addition, all proteins measured bind in a more-or-less co-operative manner to singlestranded DNA. The binding affinities for poly(dA) decrease in the following order: Y61H > wildtype > F68L and R16H > Y41F and Y41H > F73L > R21C > Y34H > G18D/Y56H. Possible explanations for the observed differences are discussed. The conservation of binding affinity, also for mutations in the single-stranded DNA-binding domain, suggests that the binding to homopolynucleotides is largely non-specific.The gene-V-encoded protein (gVp) of the F-specific Escherichia coli bacteriophage (M13, fd, fl) is a relatively small (87 amino acids) single-stranded (ss) DNA-binding protein. 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]. They also deduced a model for the gVp . ssDNA complex [15,16]. According to this model, two DNA strands bind in an antiparallel fashion to one dimer with a stoichiometry of 5 nucleotides/ protein monomer. 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. 'H-NMR binding studies of wild-type gVp with oligonucleotides containing the covalently attached spin label 4-hydroxy-1-oxyl-2,2,6,6-tetramethyl piperidine (TEMPO), have been used to locate the DNA-binding domains in ...

The DNA binding domain of the single-stranded DNA binding protein gene V protein encoded by the bacteriophage M13 was studied by means of 1H nuclear magnetic resonance, through use of a spin-labeled deoxytrinucleotide. The paramagnetic relaxation effects observed in the 1H-NMR spectrum of M13 GVP upon binding of the spin-labeled ligand were made manifest by means of 2D difference spectroscopy. In this way, a vast data reduction was accomplished which enabled us to check and extend the analysis of the 2D spectra carried out previously as well as to probe the DNA binding domain and its surroundings. The DNA binding domain is principally situated on two beta-loops. The major loop of the two is the so-called DNA binding loop (residues 16-28) of the protein where the residues which constitute one side of the beta-ladder (in particular, residues Ser20, Tyr26, and Leu28) are closest to the DNA spin-label. The other loop is part of the so-called dyad domain of the protein (residues 68-78), and mainly its residues at the tip are affected by the spin-label (in particular, Phe73). In addition, a part of the so-called complex domain of the protein (residues 44-51) which runs contiguous to the DNA binding loop is in close vicinity to the DNA. The NMR data imply that the DNA binding domain is divided over two monomeric units of the GVP dimer in which the DNA binding loop and the tip of the dyad loop are part of opposite monomers. The view of the GVP-ssDNA binding interaction which emerges from our data differs from previous molecular modeling proposals which were based on the GVP crystal structure (Brayer & McPherson, 1984; Hutchinson et al., 1990). These models implicate the involvement of one or two tyrosines (Tyr34, Tyr41) of the complex loop of the protein to participate in complex formation with ssDNA. In the NMR studies with the spin-labeled oligonucleotides, no indication of such interactions has been found. Other differences between the models and our NMR data are related to the structural differences found when solution and crystal structures are compared.