Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids. III. Binding properties of two specific proteolytic digestion products of the protein (G32P*I and G32P*III)

Nils Lonberg, Stephen C. Kowalczykowski, Leland S. Paul, Peter H. von Hippel

Research output: Contribution to journalArticle

74 Citations (Scopus)

Abstract

Brief treatment of gene 32 protein with proteolytic enzymes produces two specific digestion products in good yield (Moise & Hosoda, 1976). One, representing the native protein with ~60 amino acid residues removed from the C-terminus, is G32P*I. The other, for which ~20 amino acid residues have been removed from the N-terminus in addition to the 60 residues from the C-terminus, is G32P*III. Both of these specific "core" fragments of gene 32 protein have been isolated and purified, and their binding properties to single-stranded oligo- and polynucleotides have been studied. We find that the binding properties of G32P*I are relatively little changed from those characteristic of the native gene 32 protein: (1) the apparent binding constants to short (l = 2 to 8) oligonucleotides are independent of lattice length and essentially independent of base and sugar composition, but do show an increased salt dependence of binding relative to that of the native protein; (2) the intrinsic association constants (K) for polynucleotides binding in the co-operative mode show the same binding specificities as seen with the native protein, but with absolute values increased two to fourfold; (3) the polynucleotide binding co-operativity parameter (ω-2 × 103) and the binding site size (n ∼-7 nucleotide residues) are the same as for the native protein; (4) essentially the entire salt dependence of the net affinity (Kω) remains in K. However, unlike native gene 32 protein, G32P*I can melt native DNA to equilibrium (Hosoda et al., 1974; Greve et al., 1978); this suggests that the kinetic pathways for DNA melting by these two species must differ, since the changes in equilibrium binding parameters measured here are far too small to account for the differences in melting behavior. In contrast to G32P*I, for G32P*III we find that: (1) binding is non-cooperative (ω ∼-1); (2) the binding site size (n) for the protein has decreased by one to two nucleotide residues relative to that characteristic of the native protein and G32P*I; (3) binding to short (l = 2 to 8) oligonucleotides is length and salt concentration dependent; (4) while binding to polynucleotides continues to show approximately the same base composition dependence as the native protein, the absolute values of K are somewhat different and the salt concentration dependencies of K are less. Polynucleotide ultraviolet light and circular dichroism spectra obtained in the presence of G32P*I and G32P*III are indistinguishable from those measured with the native protein at similar binding densities, indicating that all three protein species distort the polynucleotide lattice to comparable extents. These results are combined with the equilibrium binding data for native gene 32 protein (Kowalczykowski et al., 1980a: Newport et al., 1980) to obtain further insight into the molecular details of the interactions of this protein with its nucleic acid binding substrates.

Original languageEnglish (US)
Pages (from-to)123-138
Number of pages16
JournalJournal of Molecular Biology
Volume145
Issue number1
DOIs
StatePublished - Jan 5 1981
Externally publishedYes

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Bacteriophage T4
Nucleic Acids
Proteolysis
Polynucleotides
Proteins
Salts
Oligonucleotides
Base Composition
Nucleotides
Binding Sites
Nucleic Acid Denaturation
Amino Acids
Ultraviolet Rays
Circular Dichroism

ASJC Scopus subject areas

  • Virology

Cite this

Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids. III. Binding properties of two specific proteolytic digestion products of the protein (G32P*I and G32P*III). / Lonberg, Nils; Kowalczykowski, Stephen C.; Paul, Leland S.; von Hippel, Peter H.

In: Journal of Molecular Biology, Vol. 145, No. 1, 05.01.1981, p. 123-138.

Research output: Contribution to journalArticle

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abstract = "Brief treatment of gene 32 protein with proteolytic enzymes produces two specific digestion products in good yield (Moise & Hosoda, 1976). One, representing the native protein with ~60 amino acid residues removed from the C-terminus, is G32P*I. The other, for which ~20 amino acid residues have been removed from the N-terminus in addition to the 60 residues from the C-terminus, is G32P*III. Both of these specific {"}core{"} fragments of gene 32 protein have been isolated and purified, and their binding properties to single-stranded oligo- and polynucleotides have been studied. We find that the binding properties of G32P*I are relatively little changed from those characteristic of the native gene 32 protein: (1) the apparent binding constants to short (l = 2 to 8) oligonucleotides are independent of lattice length and essentially independent of base and sugar composition, but do show an increased salt dependence of binding relative to that of the native protein; (2) the intrinsic association constants (K) for polynucleotides binding in the co-operative mode show the same binding specificities as seen with the native protein, but with absolute values increased two to fourfold; (3) the polynucleotide binding co-operativity parameter (ω-2 × 103) and the binding site size (n ∼-7 nucleotide residues) are the same as for the native protein; (4) essentially the entire salt dependence of the net affinity (Kω) remains in K. However, unlike native gene 32 protein, G32P*I can melt native DNA to equilibrium (Hosoda et al., 1974; Greve et al., 1978); this suggests that the kinetic pathways for DNA melting by these two species must differ, since the changes in equilibrium binding parameters measured here are far too small to account for the differences in melting behavior. In contrast to G32P*I, for G32P*III we find that: (1) binding is non-cooperative (ω ∼-1); (2) the binding site size (n) for the protein has decreased by one to two nucleotide residues relative to that characteristic of the native protein and G32P*I; (3) binding to short (l = 2 to 8) oligonucleotides is length and salt concentration dependent; (4) while binding to polynucleotides continues to show approximately the same base composition dependence as the native protein, the absolute values of K are somewhat different and the salt concentration dependencies of K are less. Polynucleotide ultraviolet light and circular dichroism spectra obtained in the presence of G32P*I and G32P*III are indistinguishable from those measured with the native protein at similar binding densities, indicating that all three protein species distort the polynucleotide lattice to comparable extents. These results are combined with the equilibrium binding data for native gene 32 protein (Kowalczykowski et al., 1980a: Newport et al., 1980) to obtain further insight into the molecular details of the interactions of this protein with its nucleic acid binding substrates.",
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N2 - Brief treatment of gene 32 protein with proteolytic enzymes produces two specific digestion products in good yield (Moise & Hosoda, 1976). One, representing the native protein with ~60 amino acid residues removed from the C-terminus, is G32P*I. The other, for which ~20 amino acid residues have been removed from the N-terminus in addition to the 60 residues from the C-terminus, is G32P*III. Both of these specific "core" fragments of gene 32 protein have been isolated and purified, and their binding properties to single-stranded oligo- and polynucleotides have been studied. We find that the binding properties of G32P*I are relatively little changed from those characteristic of the native gene 32 protein: (1) the apparent binding constants to short (l = 2 to 8) oligonucleotides are independent of lattice length and essentially independent of base and sugar composition, but do show an increased salt dependence of binding relative to that of the native protein; (2) the intrinsic association constants (K) for polynucleotides binding in the co-operative mode show the same binding specificities as seen with the native protein, but with absolute values increased two to fourfold; (3) the polynucleotide binding co-operativity parameter (ω-2 × 103) and the binding site size (n ∼-7 nucleotide residues) are the same as for the native protein; (4) essentially the entire salt dependence of the net affinity (Kω) remains in K. However, unlike native gene 32 protein, G32P*I can melt native DNA to equilibrium (Hosoda et al., 1974; Greve et al., 1978); this suggests that the kinetic pathways for DNA melting by these two species must differ, since the changes in equilibrium binding parameters measured here are far too small to account for the differences in melting behavior. In contrast to G32P*I, for G32P*III we find that: (1) binding is non-cooperative (ω ∼-1); (2) the binding site size (n) for the protein has decreased by one to two nucleotide residues relative to that characteristic of the native protein and G32P*I; (3) binding to short (l = 2 to 8) oligonucleotides is length and salt concentration dependent; (4) while binding to polynucleotides continues to show approximately the same base composition dependence as the native protein, the absolute values of K are somewhat different and the salt concentration dependencies of K are less. Polynucleotide ultraviolet light and circular dichroism spectra obtained in the presence of G32P*I and G32P*III are indistinguishable from those measured with the native protein at similar binding densities, indicating that all three protein species distort the polynucleotide lattice to comparable extents. These results are combined with the equilibrium binding data for native gene 32 protein (Kowalczykowski et al., 1980a: Newport et al., 1980) to obtain further insight into the molecular details of the interactions of this protein with its nucleic acid binding substrates.

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