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

Abstract

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 ...

Structure of the gene V protein of bacteriophage f1 determined by multiwavelength x-ray diffraction on the selenomethionyl protein.

“…The structure of the protein-ssDNA complex has been studied using electron microscopy and solution scattering methods and is found to consist of a regular left-handed superhelix in which the gene V protein dimers are arrayed on the outside of the superhelix and the ssDNA strands are inside (8,25). The structure of the gene V protein is also of interest because its small size and the large number of mutants available have made it a useful model for determining effects ofamino acid substitutions on protein stability and function (23,26,27).A model for the crystal structure of the wild-type (WT) gene V protein has been reported (28), but recent NMR studies have demonstrated that the positions of amino acids involved in the segments of antiparallel (-structure are not compatible with those in the model (21), and a new determination of the structure was necessary. The multiwavelength anomalous diffraction (MAD) technique (29) was ideally suited for this purpose, as the WT gene V protein contains two methionine residues (Met-1 and Met-77) that might be substituted in vivo in Escherichia coli by selenomethionine.…”

“…Gene V protein binding to single-stranded nucleic acids and to oligonucleotides has been studied using chemical modification, spectroscopic techniques, and mutagenesis (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). The structure of the protein-ssDNA complex has been studied using electron microscopy and solution scattering methods and is found to consist of a regular left-handed superhelix in which the gene V protein dimers are arrayed on the outside of the superhelix and the ssDNA strands are inside (8,25).…”

“…The structure of the protein-ssDNA complex has been studied using electron microscopy and solution scattering methods and is found to consist of a regular left-handed superhelix in which the gene V protein dimers are arrayed on the outside of the superhelix and the ssDNA strands are inside (8,25). The structure of the gene V protein is also of interest because its small size and the large number of mutants available have made it a useful model for determining effects ofamino acid substitutions on protein stability and function (23,26,27).…”

Analyses of the stability and function of three surface mutants (R82C, K69H, and L32R) of the gene V protein from Ff phage by X‐ray crystallography

“…In particular, the participation of Y41 in the protein dimer-dimer contacts in the GVP-ssDNA complex (King & Coleman, 1988;Stassen et al, 1992) was explained adequately. A similar model has been proposed independently using the three-dimensional structure of Y41H GVP determined by NMR Folmer et al, 1994).…”

“…During the rolling circle replication process, GVP binds to the ssDNA cooperatively and, ultimately, a superhelical GVP-DNA complex is formed. The structure of such helical complexes has been studied by a number of biophysical methods, including electron microscopy (Gray, 1989;Olah et al, 1995), neutron scattering (Gray et al, 1982;Olah et al, 1995), NMR and fluorescence spectroscopies (King & Coleman, 1987Dick et al, 1989;Stassen et al, 1992;Folkers et al, 1993). The results of these studies have provided valuable insights regarding the interactions of GVP and ssDNA.…”