Specificity of the Binding of Bacteriophage M13 Encoded Gene-5 Protein to DNA and RNA Studied by Means of Fluorescence Titrations

Bulsink, Henk; Harmsen, B.J.M.; Hilbers, Cornelis W.

doi:10.1080/07391102.1985.10508413

Cited by 63 publications

(90 citation statements)

References 53 publications

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“…on the initial degree of saturation. This is observed for the M13 DNA complexes: [15]. It indicates that the salt concentration mainly influences the association rate: d log k,/d log [NaCI] = -3.32.…”

Section: Discussionmentioning

confidence: 75%

DNA‐binding properties of gene‐5 protein encoded by bacteriophage M 13

Bulsink

Harmsen

Hilbers

1988

European Journal of Biochemistry

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The irreversible dissociation kinetics of complexes of M13-encoded gene-5 protein with the polynucleotides poly(dA) and M13 DNA was studied by means of stopped-flow experiments. A linear decay was found for all gene-5-protein . poly(dA) complexes and for the gene-5-protein . M13 DNA complexes for which the DNA lattice was completely saturated at the beginning of the dissociation experiments. Only at the end of the dissociation curve was a deviation from linearity observed. A single-exponential decay was found for the dissociation of gene-5-protein . M13 DNA complexes when the DNA was not completely saturated initially. These results could be interpreted by assuming that dissociation of bound protein is only possible from isolated binding sites, while during the dissociation, rearrangement of bound protein clusters takes place continuously, including the formation of newly isolated bound protein. This redistribution results from a translocation of the protein along the lattice, which, for the poly(dA) complex, is fast with respect to the dissociation step, but which is slow for the M13 DNA complex. During this process the equilibrium cluster distribution predicted by the theory of McGhee and Von Hippel [I] is not maintained.The binding of gene-5 protein to poly(dA) or poly(dT) does not result in a broadening of the nucleotide resonances in the NMR spectra of these polynucleotides, as had been observed for E. coli DNA-binding protein and interpreted as an indication for a high rate of translocation of the protein on the polynucleotide [2]. The absence of line broadening for gene-5-protein . polynucleotide complexes is caused by the high binding cooperativity. As a consequence the majority of the protein molecules are bound in a cluster which makes the concentration of isolated bound protein very low. This results in a decrease of the signal/noise ratio at higher degrees of binding, but does not lead to line broadening while fast translocation still occurs.

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Section: Discussionmentioning

confidence: 75%

DNA‐binding properties of gene‐5 protein encoded by bacteriophage M 13

Bulsink

Harmsen

Hilbers

1988

European Journal of Biochemistry

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“…Each subunit of protein is part of a dimer, and the two subunits in a dimer bind simultaneously to two separate strands of nucleic acid. titrations of gene V protein-poly(dA) complexes were interpreted by Bulsink et al (1985) in terms of a value of ω of about 500. Titrations of poly(dA) with gene V protein at concentrations of salt ranging from 0.11 to 0.22 M NaCl were carried out by Alma et al (1983), yielding estimates of ω ranging from 50 to 290, with no clear dependence on salt.…”

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confidence: 99%

Gene V Protein Dimerization and Cooperativity of Binding to Poly(dA)

Terwilliger

1996

Biochemistry

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Gene V protein of bacteriophage f1 is a dimeric protein that binds cooperatively to single-stranded nucleic acids. In order to determine whether a monomer-dimer equilibrium has an appreciable effect upon the thermodynamics of gene V protein binding to nucleic acids, the dissociation constant for the protein dimer was investigated using size-exclusion chromatography. At concentrations ranging from 5 x 10(-10) to 1.2 x 10(-5) M, the Stokes radius of the protein was that expected of the dimer of the gene V protein. The Stokes radius of the protein was also independent of salt concentration from 0.2 to 1.0 M NaCl in a buffer containing 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA. The binding of the dimeric gene V protein to poly(dA) was studied using a simplified lattice model for protein-protein interactions adapted for use with a dimeric protein that binds simultaneously to two strands of nucleic acid. Interpretation of the salt dependence, C = [d log(Kint omega)]/[d log(NaCl)], of binding of such a dimeric protein to nucleic acid using the theory of Record et al. (Record, M. T., et al. (1976) J. Mol. Biol. 107, 145-158) indicates that C is a function of the numbers of cations and anions released from protein and nucleic acid upon binding of the dimer, not of the monomer. Cooperativity of gene V protein binding to poly(dA) was studied with titration experiments that are sensitive to the degree of cooperativity of binding. The cooperativity factor omega, defined as the ratio of the binding constant for a site adjacent to a previously bound dimer to that for an isolated site, was found to be relatively insensitive to salt, with a value in the range of 2000-7000 for binding to poly(dA) at 3 degrees C and at 23 degrees C. This high cooperativity factor supports the suggestion that protein-protein contacts play a major role in the formation of the superhelical gene V protein-single-stranded nucleic acid complex.

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“…This fluorescence is quenched as the protein binds to ssDNA and is restored when the proteinssDNA complex is dissociated by the addition of NaCl. Binding affinities of WT gene V protein to a variety of substrates and of WT and several mutant gene V proteins to the substrate polydeoxyadenylic acid have been reported (31,(33)(34)(35)(36). Thus the gene V protein provides a system in which proteins containing amino acid substitutions may be readily obtained and characterized in vitro.…”

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confidence: 99%

Engineering multiple properties of a protein by combinatorial mutagenesis.

Sandberg

Terwilliger

1993

Proc. Natl. Acad. Sci. U.S.A.

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A method for simultaneously engineering multiple properties of a protein, based on the observed additivity of effects of individual mutations, b presented. We show that, for the gene V protein of bacteriophage n, effects of double mutations on both protein stability and DNA binding ainity are approximately equal to the sums ofthe effects ofthe consituent single mutations. This additivity of effects implies that it Is posible to deliberately construct mutant proteins optimizd for multiple properties by combination of appropriate single mutations chosen from a characterized library.One of the long-term goals of the study of mutational effects on protein stability and activity is to devise a method by which mutations can be rationally employed to alter or "engineer" the properties of proteins with predictable results. Recombinant DNA technology has allowed the construction of proteins of altered stability in vitro (1-16), catalytic efficiency (17-19), substrate specificity (20-23), and resistance to in vivo thermal inactivation (24) through the use of single or multiple amino acid substitutions. This effort has been greatly helped by the fact that the effects of amino acid substitutions on such properties of proteins tend to be additive as mutations accumulate, provided that the substituting residues do not interact functionally or by direct contact (25). For example, additive increases in the stability of subtilisin BPN' have been achieved by combining mutations at six sites in the protein tertiary structure (16). The six mutations individually stabilize the protein by 0.3-1.3 kcal/ mol, and the individual effects sum to a stability increase of 3.8 kcal/mol predicted for the hexa-mutant. The observed stabilization of the mutant containing all six substitutions is 4.3 kcal/mol (16). Additive effects ofamino acid substitutions have been used to engineer incremental increases in the stability of other proteins including the N-terminal domain of A repressor (5, 13), T4 lysozyme (9, 12), kanamycin nucleotidyltransferase (6), and neutral protease (26) as well. This strategy of additive mutation has also been employed to alter binding affinities or specificities of proteins, such as A repressor (20), subtilisin (21-23), and glutathione reductase (27), for their substrates or cofactors, and to alter the pH profile of subtilisin (17).A factor complicating the effort to engineer proteins by mutation is that most single amino acid substitutions alter multiple properties ofthe proteins in which they are made. To be functional, a protein must be at once stable, yet flexible, with high catalytic activity balanced against substrate specificity. Because mutants affecting only one of these properties are relatively rare, it appears difficult to optimize one characteristic of a protein through mutations while maintaining adequacy in the others. However, the observation that mutational effects on the in vitro properties of proteins areThe publication costs of this article were defrayed in part by page charge payment. This ar...

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Specificity of the Binding of Bacteriophage M13 Encoded Gene-5 Protein to DNA and RNA Studied by Means of Fluorescence Titrations

Cited by 63 publications

References 53 publications

DNA‐binding properties of gene‐5 protein encoded by bacteriophage M 13

DNA‐binding properties of gene‐5 protein encoded by bacteriophage M 13

Gene V Protein Dimerization and Cooperativity of Binding to Poly(dA)

Engineering multiple properties of a protein by combinatorial mutagenesis.

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