title: DNA-binding properties of gene-5 protein encoded by bacteriophage M13. 2. Further characterization of the different binding modes for poly- and oligodeoxynucleic acids.
abstract: 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.
authors.affiliation: Laboratory of Biophysical Chemistry, University of Nijmegen, The Netherlands.
authors.forename: H
authors.initials: H
authors.lastname: Bulsink
authors.forename: B J
authors.initials: BJ
authors.lastname: Harmsen
authors.forename: C W
authors.initials: CW
authors.lastname: Hilbers
mesh_terms: D001665:Binding Sites; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D009904:Optical Rotation; D011485:Protein Binding; D012965:Sodium Chloride; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D012965:Sodium Chloride