DNA-binding properties of gene-5 protein encoded by bacteriophage M13. 2. Further characterization of the different binding modes for poly- and oligodeoxynucleic acids

The binding of gene-5 protein, encoded by bacteriophage M13, to oligodeoxynucleic acids was studied by means of fluorescence binding experiments, fluorescence depolarization measurements and irreversible dissociation kinetics of the protein.nucleotide complexes with salt. The binding properties thus obtained are compared with those of the binding to polynucleotides, especially at very low salt concentration. It appears that the binding to oligonucleotides is always characterized by a stoichiometry (n) of 2-3 nucleotides/protein, and the absence of cooperativity. In contrast the protein can bind to polynucleotides in two different modes, one with a stoichiometry of n = 3 in the absence of salt and another with n = 4 at moderate salt concentrations. Both modes have a high intramode cooperativity (omega about 500) but are non-interacting and mutually exclusive. For deoxynucleic acids with a chain length of 25-30 residues a transition from oligonucleotide to polynucleotide binding is observed at increasing nucleotide/protein ratio in the solution. The n = 3 polynucleotide binding is very sensitive to the ionic strength and is only detectable at very low salt concentrations. The ionic strength dependency per nucleotide of the n = 4 binding is much less and is comparable with the salt dependency of the oligonucleotide binding. Furthermore it appears that the influence of the salt concentration on the oligonucleotide binding constant is to about the same degree determined by the effect of salt on the association and dissociation rate constants. Model calculations indicate that the fluorescence depolarization titration curves can only be explained by a model for oligonucleotide binding in which a protein dimer binds with its two dimer halves to the same strand. In addition it is only possible to explain the observed effect of the chain length of the oligonucleotide on both the apparent binding constant and the dissociation rate by assuming the existence of interactions between protein dimers bound to different strands. This results in the formation of a complex consisting of two nucleotide strands with protein in between and stabilized by the dimer-dimer interactions.     

Specificity of the binding of bacteriophage M13 encoded gene-5 protein to DNA and RNA studied by means of fluorescence titrations

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.     

Time-resolved fluorescence of bacteriophage Pf1 DNA-binding protein. Determination of oligonucleotide and polynucleotide binding parameters

The binding of oligonucleotides and polynucleotides to the Pf1 DNA-binding protein was followed by fluorescence spectral shift and lifetime measurements, which gave an anomalous value for the stoichiometry of binding. The anomaly was investigated in detail using fluorescence depolarisation to measure the aggregation during the titration and showed that all the fluorescence parameters are related to the specific aggregation of dimers on ligand binding. At saturation, complexes of the protein with the octanucleotide d(GCGTTGCG) and the hexadecanucleotide (dT)16 have rotational correlation times, phi, of 50 ns and 85 ns, corresponding to protein tetramers and octamers, respectively. In the presence of the tetranucleotide d(CGCA) the protein remains as the native dimer (phi = 19 ns). The titration curves could be analysed in terms of two non-equivalent binding sites, with binding constants K1 and K2. Comparison of K1 values for oligonucleotide binding leads to an estimated (single-site) intrinsic binding constant Kint approximately equal to 3 X 10(4) M-1 and a cooperativity parameter omega approximately equal to 100, in agreement with the apparent binding constant Kapp approximately equal to 3 X 10(6) M-1 for polynucleotides. Binding to the second site on the protein dimer is greatly reduced and cannot be determined accurately. The results suggest that the protein dimers bind cooperatively by lateral association along the DNA and that occupation of only one of the two DNA-binding sites of the protein dimers is sufficient to stabilize the nucleoprotein complexes.     

Complex of bacteriophage M13 single-stranded DNA and gene 5 protein

Lysates of bacteriophage M13-infected cells contain numerous unbranched filamentous structures approximately 1·1 μm long × 160 Å wide, that is, slightly longer and considerably wider than M13 virions. These structures are complexes of viral single-stranded DNA molecules with M13 gene 5 protein, a non-capsid protein required for single-stranded DNA production. All, or nearly all, of the single-stranded DNA from the infected cells and at least half to two-thirds of the gene 5 protein molecules are found as complex in the lysates. The complex contains about 1300 gene 5 protein molecules per DNA molecule but little if any of the two known capsid proteins. The complex is much less stable than virions in the presence of salt or ionic detergent solutions and in electron micrographs it appears to have a much looser and more open structure. If an excess of M13 single-stranded DNA is added to complex in a lysate, the gene 5 protein molecules from the complex redistribute onto all of the added as well as the original DNA, again suggesting a rather loose association of protein and DNA.By electron microscopy, the complex from infected cells appears to differ structurally from complex formed in vitro between purified single-stranded DNA and purified gene 5 protein. Because of this apparent structural difference and because previous experiments suggested the presence of complex in vivo, we presume that the complex which we have found in lysates of infected cells previously did exist as such inside the cells, but we have been unable to exclude that it formed during or after lysis. If it is assumed that complex does occur in vivo, the results of pulse-chase radioactive labeling experiments on infected cells can be interpreted as showing that with time the single-stranded DNA leaves complex, presumably to be matured into virions, while the gene 5 protein molecules are re-used to form more complex.     

Replication of bacteriophage M13. XII. In vivo cross-linking of a phage-specific DNA binding protein to the single-stranded DNA of bacteriophage M13 by ultraviolet irradiation.

Ultraviolet irradiation of bacteriophage M13-infected Escherichia coli induces the formation of a covalent crosslink between progeny single-stranded DNA and the M13 DNA binding protein, the product of gene 5. The crosslinked complex is readily isolated from detergent-treated lysates by sucrose-gradient velocity sedimentation and CsCl equilibrium sedimentation in the presence of detergent. The crosslinked complex produced with optimal levels of irradiation sediments 1.06 times faster than uncomplexed M13 single-stranded DNA, has a buoyant density of approximately 1.62 to 1.64 g/cm³ and a protein to DNA mass ratio of 2 mg protein per mg DNA. Cleavage of the crosslinked complex with cyanogen bromide or trypsin yields products similar to those produced by cleavage of purified M13 gene 5 protein. The crosslink is located close to the carboxyl terminus of the protein.     

Inserting foreign peptides into the major coat protein of bacteriophage M13.

Foreign DNA fragments were inserted into filamentous phage gene VIII to create hybrid B-proteins with foreign sequences in the amino terminus. The hybrid proteins are incorporated into the virions which retain viability and infectivity. Virions with hybrid B-proteins have the same contour length and the same number of B-protein molecules as virions with natural B-proteins. It was shown that for one of hybrid B-proteins the position of the processing site had changed.     

Characterization of two conformational forms of the major DNA-binding protein encoded by herpes simplex virus 1.

We have resolved two electrophoretic species of the major DNA-binding protein, infected cell polypeptide 8 (ICP8), encoded by herpes simplex virus 1. In pulse-chase experiments, we observed the conversion of the ICP8a form, the slower migrating species, to the faster migrating form, ICP8b. Thus, the two species appear to be related as precursor-product. The conversion was not due to proteolytic cleavage, because higher concentrations of reducing agents in the sample buffer shifted the faster moving form to the slower moving species. Also, the two forms have identical peptide patterns as analyzed by partial proteolysis in sodium dodecyl sulfate. Thus, the faster moving species appears to be a conformational isomer containing intramolecular disulfide bonds. The functional significance of the two forms of the protein is discussed.     

Comprehensive mutagenesis of the C-terminal domain of the M13 gene-3 minor coat protein: the requirements for assembly into the bacteriophage particle

Filamentous bacteriophage assemble at the host membrane in a non-lytic process; the gene-3 minor coat protein (P3) is required for release from the membrane and subsequently, for recognition and infection of a new host. P3 contains at least three distinct domains: two N-terminal domains that mediate host recognition and infection, and a C-terminal domain (P3-C) that is required for release from the host cell following phage assembly and contributes to the structural stability of the phage particle. A comprehensive mutational analysis of the 150 residue P3-C revealed that only 24 side-chains, located within the last 70 residues of sequence, were necessary for efficient incorporation into a wild-type coat. The results reveal that the requirements for the assembly of P3 into the phage particle are quite lax and involve only a few key side-chains. These findings shed light on the functional and structural requirements for filamentous phage assembly, and they may provide guidelines for the engineering of improved coat proteins as scaffolds for phage display technology.     

Fluorescence studies of the complex formation between the gene 5 protein of bacteriophage M13 and polynucleotides

The binding characteristics of the interaction of gene 5 protein with polynucleotides, i.e. poly(dA), poly(dT) and M13 DNA, have been determined by following the quenching of the protein fluorescence. In general, the binding is highly co-operative and for the binding of the protein to poly(dA) and M13 DNA the co-operativity parameter ω is estimated to have values between 50 and 300. Under comparable experimental conditions, the intrinsic binding constant K_int is at least two orders of magnitude higher for poly(dT) than for poly(dA), while the value for M13 DNA is intermediate. For poly(dA), the binding has been studied as a function of ionic strength and temperature. From these experiments it can be concluded that ionic interactions as well as van der Waals interactions (e.g. stacking interactions) are important for the complex formation of the protein with polynucleotides. From a comparison of the binding of the protein to poly(dA) and poly(dT), it is concluded that stacking interactions in the polynucleotide have a negative influence on protein binding. This conclusion, in conjunction with the weak temperature dependence of K_int. indicates that ionic interactions play a major role in the stabilization of the protein-poly(dA) complex. The co-operativity factor ω is little or not dependent on the ionic strength or the type of polynucleotide involved in binding. It is determined by interactions between complexed protein molecules. These interactions are primarily non-electrostatic.The binding characteristics obtained for the gene 5 protein-polynucleotide complexes are compared with those we have found for the binding to small oligonucleotides. It appears that oligonucleotide and polynucleotide binding differ in many aspects; i.e. there is a difference in K_int, ω and the number of nucleotides covered. The validity of linear lattice binding theories is discussed in this context. By comparing the binding parameters found for the gene 5 protein with those of the Escherichia coli DNA binding protein I. it is possible to explain the displacement of the E. coli protein by the gene 5 protein that occurs in vivo.     

Purification and DNA-binding properties of the cro-type regulatory repressor protein cng encoded by the Lactobacillus plantarum phage phi g1e

The putative repressor protein Cng (10kDa on an SDS gel) for the lytic pathway of Lactobacillus plantarum phage φg1e was purified using the Escherichia coli Pt7 system, and its DNA-binding ability for the seven operator-like sequences, the GATAC-boxes (Gb1 to Gb7), was investigated in vitro. In gel-shift assays, Cng selectively bound to the DNA fragments containing the GATAC-box(es). In addition, DNase I footprinting analysis with supercoiled DNA demonstrated that Cng can specifically cover about a 25bp region centered around each of the GATAC-boxes, although two boxes, Gb4 and Gb6, were only partially protected. Moreover, protein crosslinking experiments using glutaraldehyde suggested that Cng most likely functions as a dimer. On the other hand, the binding ability of Cpg for the GATAC-boxes in supercoiled DNA was also examined under the same conditions as in Cng; unlike Cng, Cpg covered Gb4 and Gb6 completely sufficiently as well as the other five boxes. Thus, the present and previous [Kakikawa et al., Gene 215 (1998) 371-379; 242 (2000) 155-166] results indicate a possibility that the two proteins Cng and Cpg selectively bind to the GATAC-boxes that act as operators, and can decide between the lytic or lysogenic pathways through repression of the promoter activity of P(R) as well as P(L).     

Spin-label ESR of bacteriophage M13 coat protein in mixed lipid bilayers. Characterization of molecular selectivity of charged phospholipids for the bacteriophage M13 coat protein in lipid bilayers

Bacteriophage M13 major coat protein has been incorporated at different lipid/protein ratios in lipid bilayers consisting of various ratios of dimyristoylphosphatidylcholine (DMPC) to dimyristoylphosphatidylglycerol (DMPG). Spin-label ESR experiments were performed with phospholipids labeled at the C-14 position of the sn-2 chain. For M13 coat protein recombinants with DMPC alone, the relative association constants were determined for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels and found to be 1.0, 1.0, and 2.1 relative to the background DMPC, respectively. The number of association sites for each phospholipid on the protein was found to be 4 per protein monomer. The intrinsic off-rates for lipid exchange at the intramembranous surface of the protein in DMPC alone at 30 degrees C were found to be 5 X 10(6), 6 X 10(6), and 2 X 10(6) s-1 for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels, respectively. Adding DMPG to the DMPC lipid system increased the exchange rates of the lipids on and off the protein. By gel filtration chromatography, it is found that protein aggregation is reduced after addition of DMPG to the lipid system. This is in agreement with measurements of tryptophan fluorescence, which show a decrease in quenching efficiency after introduction of DMPG in the lipid system. The results are interpreted in terms of a model relating the ESR data to the size of the protein-lipid aggregates.