Equilibrium binding of Escherichia coli single-strand binding protein to single-stranded nucleic acids in the (SSB)65 binding mode. Cation and anion effects and polynucleotide specificity

Leslie B. Overman, Wlodzimierz Bujalowski, Timothy M. Lohman

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Abstract

The Escherichia coli single-strand binding (SSB) protein binds single-stranded (ss) nucleic acids in at least four distinct binding modes depending on the salt conditions [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799]. Equilibrium binding constants for the interaction of the E. coli SSB protein with poly(A), poly(U), poly(dA), and poly(dT) have been measured over a range of monovalent salt concentrations and types under conditions which favor only the high site size, (SSB)65 binding mode, which covers 65 nucleotides per SSB tetramer. The binding isotherms are analyzed by using a statistical thermodynamic model ("tetramer/octamer" model) that assumes cooperative binding of SSB is limited to the formation of octamers [Bujalowski, W., & Lohman, T. M. (1987) J. Mol. Biol. 195, 897] rather than the indefinite clustering of tetramers. The dependence of the intrinsic association equilibrium constant, Kobsd, and cooperatively parameter, ωT/O, on salt concentration has been determined by titrations which monitor the fluorescence quenching of the SSB protein upon complex formation. In the (SSB)65 binding mode, SSB binds with only moderate cooperativity to ss nucleic acids [Lohman, T. M., Overman, L. B., & Datta, S. (1986) J. Mol. Biol. 187, 603]. The cooperativity parameter derived from the tetramer/octamer model, which represents the equilibrium constant for formation of a nucleic acid bound SSB octamer from two nucleic acid bound tetramers, has a value of ωT/O = 410 ± 120 and is independent of salt concentration and type for poly(dA), poly(U), and poly(A) (25°C, pH 8.1). However, Kobsd decreases steeply with increasing salt concentration, such that ∂ log Kobsd/∂ log [NaCl] = -7.4 ± 0.5 for poly(U), -6.1 ± 0.6 for poly(dA), and -6.2 ± 0.3 for poly(A) (25.0°C, pH 8.1). The SSB-poly(dT) affinity is too high to measure in buffers containing even 5 M NaCl; however, in 1.8-2.5 M NaBr, we measure ∂ log Kobsd/∂ log [NaBr] = -5.7 ± 0.7, with a lower value of ωT/O = 130 ± 70. The polynucleotide specificity of the (SSB)65 binding mode (0.20 M NaCl, 25.0°C, pH 8.1) is Kobsd(dT) > Kobsd(dC) ≫ Kobsd(ss M13 DNA) > Kobsd(I) > Kobsd(U) = 8Kobsd(dA) = 87Kobsd(A) ≫ Kobsd(C). A dramatic effect of anion type on both the salt dependence and magnitude of Kobsd is also observed such that for poly(U) ∂ log Kobsd/∂ log [potassium glutamate] = -5.7 ± 0.4, ∂ log Kobsd/∂ log [NaF] = -4.3 ± 0.4, ∂ log Kobsd/∂ log [NaCH3COO] = -6.5 ± 0.2, and ∂ log Kobsd/∂ log [NaBr] = -6.7 ± 0.6; in 0.35 M monovalent cation, Kobsd(Glu) = 5.6Kobsd(F) = 11Kobsd(CH3COO) = (1.1 × 103)Kobsd(Cl) = (1.1 × 104)Kobsd(Br). These data indicate that significant electrostatic interactions occur in the (SSB)65 complexes formed with all poly-nucleotides, resulting in a net release of both cations and anions, although there are also contributions due to cation and anion uptake.

Original languageEnglish (US)
Pages (from-to)456-471
Number of pages16
JournalBiochemistry
Volume27
Issue number1
StatePublished - 1988
Externally publishedYes

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Polynucleotides
Escherichia coli
Nucleic Acids
Anions
Poly U
Cations
Carrier Proteins
Salts
Poly A
Equilibrium constants
Nucleotides
Poly A-U
Poly T
Monovalent Cations
Biochemistry
Statistical mechanics
Single-Stranded DNA
Statistical Models
Coulomb interactions
Static Electricity

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{8c251cc3b80c421cb018dbc6d77cfe52,
title = "Equilibrium binding of Escherichia coli single-strand binding protein to single-stranded nucleic acids in the (SSB)65 binding mode. Cation and anion effects and polynucleotide specificity",
abstract = "The Escherichia coli single-strand binding (SSB) protein binds single-stranded (ss) nucleic acids in at least four distinct binding modes depending on the salt conditions [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799]. Equilibrium binding constants for the interaction of the E. coli SSB protein with poly(A), poly(U), poly(dA), and poly(dT) have been measured over a range of monovalent salt concentrations and types under conditions which favor only the high site size, (SSB)65 binding mode, which covers 65 nucleotides per SSB tetramer. The binding isotherms are analyzed by using a statistical thermodynamic model ({"}tetramer/octamer{"} model) that assumes cooperative binding of SSB is limited to the formation of octamers [Bujalowski, W., & Lohman, T. M. (1987) J. Mol. Biol. 195, 897] rather than the indefinite clustering of tetramers. The dependence of the intrinsic association equilibrium constant, Kobsd, and cooperatively parameter, ωT/O, on salt concentration has been determined by titrations which monitor the fluorescence quenching of the SSB protein upon complex formation. In the (SSB)65 binding mode, SSB binds with only moderate cooperativity to ss nucleic acids [Lohman, T. M., Overman, L. B., & Datta, S. (1986) J. Mol. Biol. 187, 603]. The cooperativity parameter derived from the tetramer/octamer model, which represents the equilibrium constant for formation of a nucleic acid bound SSB octamer from two nucleic acid bound tetramers, has a value of ωT/O = 410 ± 120 and is independent of salt concentration and type for poly(dA), poly(U), and poly(A) (25°C, pH 8.1). However, Kobsd decreases steeply with increasing salt concentration, such that ∂ log Kobsd/∂ log [NaCl] = -7.4 ± 0.5 for poly(U), -6.1 ± 0.6 for poly(dA), and -6.2 ± 0.3 for poly(A) (25.0°C, pH 8.1). The SSB-poly(dT) affinity is too high to measure in buffers containing even 5 M NaCl; however, in 1.8-2.5 M NaBr, we measure ∂ log Kobsd/∂ log [NaBr] = -5.7 ± 0.7, with a lower value of ωT/O = 130 ± 70. The polynucleotide specificity of the (SSB)65 binding mode (0.20 M NaCl, 25.0°C, pH 8.1) is Kobsd(dT) > Kobsd(dC) ≫ Kobsd(ss M13 DNA) > Kobsd(I) > Kobsd(U) = 8Kobsd(dA) = 87Kobsd(A) ≫ Kobsd(C). A dramatic effect of anion type on both the salt dependence and magnitude of Kobsd is also observed such that for poly(U) ∂ log Kobsd/∂ log [potassium glutamate] = -5.7 ± 0.4, ∂ log Kobsd/∂ log [NaF] = -4.3 ± 0.4, ∂ log Kobsd/∂ log [NaCH3COO] = -6.5 ± 0.2, and ∂ log Kobsd/∂ log [NaBr] = -6.7 ± 0.6; in 0.35 M monovalent cation, Kobsd(Glu) = 5.6Kobsd(F) = 11Kobsd(CH3COO) = (1.1 × 103)Kobsd(Cl) = (1.1 × 104)Kobsd(Br). These data indicate that significant electrostatic interactions occur in the (SSB)65 complexes formed with all poly-nucleotides, resulting in a net release of both cations and anions, although there are also contributions due to cation and anion uptake.",
author = "Overman, {Leslie B.} and Wlodzimierz Bujalowski and Lohman, {Timothy M.}",
year = "1988",
language = "English (US)",
volume = "27",
pages = "456--471",
journal = "Biochemistry",
issn = "0006-2960",
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number = "1",

}

TY - JOUR

T1 - Equilibrium binding of Escherichia coli single-strand binding protein to single-stranded nucleic acids in the (SSB)65 binding mode. Cation and anion effects and polynucleotide specificity

AU - Overman, Leslie B.

AU - Bujalowski, Wlodzimierz

AU - Lohman, Timothy M.

PY - 1988

Y1 - 1988

N2 - The Escherichia coli single-strand binding (SSB) protein binds single-stranded (ss) nucleic acids in at least four distinct binding modes depending on the salt conditions [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799]. Equilibrium binding constants for the interaction of the E. coli SSB protein with poly(A), poly(U), poly(dA), and poly(dT) have been measured over a range of monovalent salt concentrations and types under conditions which favor only the high site size, (SSB)65 binding mode, which covers 65 nucleotides per SSB tetramer. The binding isotherms are analyzed by using a statistical thermodynamic model ("tetramer/octamer" model) that assumes cooperative binding of SSB is limited to the formation of octamers [Bujalowski, W., & Lohman, T. M. (1987) J. Mol. Biol. 195, 897] rather than the indefinite clustering of tetramers. The dependence of the intrinsic association equilibrium constant, Kobsd, and cooperatively parameter, ωT/O, on salt concentration has been determined by titrations which monitor the fluorescence quenching of the SSB protein upon complex formation. In the (SSB)65 binding mode, SSB binds with only moderate cooperativity to ss nucleic acids [Lohman, T. M., Overman, L. B., & Datta, S. (1986) J. Mol. Biol. 187, 603]. The cooperativity parameter derived from the tetramer/octamer model, which represents the equilibrium constant for formation of a nucleic acid bound SSB octamer from two nucleic acid bound tetramers, has a value of ωT/O = 410 ± 120 and is independent of salt concentration and type for poly(dA), poly(U), and poly(A) (25°C, pH 8.1). However, Kobsd decreases steeply with increasing salt concentration, such that ∂ log Kobsd/∂ log [NaCl] = -7.4 ± 0.5 for poly(U), -6.1 ± 0.6 for poly(dA), and -6.2 ± 0.3 for poly(A) (25.0°C, pH 8.1). The SSB-poly(dT) affinity is too high to measure in buffers containing even 5 M NaCl; however, in 1.8-2.5 M NaBr, we measure ∂ log Kobsd/∂ log [NaBr] = -5.7 ± 0.7, with a lower value of ωT/O = 130 ± 70. The polynucleotide specificity of the (SSB)65 binding mode (0.20 M NaCl, 25.0°C, pH 8.1) is Kobsd(dT) > Kobsd(dC) ≫ Kobsd(ss M13 DNA) > Kobsd(I) > Kobsd(U) = 8Kobsd(dA) = 87Kobsd(A) ≫ Kobsd(C). A dramatic effect of anion type on both the salt dependence and magnitude of Kobsd is also observed such that for poly(U) ∂ log Kobsd/∂ log [potassium glutamate] = -5.7 ± 0.4, ∂ log Kobsd/∂ log [NaF] = -4.3 ± 0.4, ∂ log Kobsd/∂ log [NaCH3COO] = -6.5 ± 0.2, and ∂ log Kobsd/∂ log [NaBr] = -6.7 ± 0.6; in 0.35 M monovalent cation, Kobsd(Glu) = 5.6Kobsd(F) = 11Kobsd(CH3COO) = (1.1 × 103)Kobsd(Cl) = (1.1 × 104)Kobsd(Br). These data indicate that significant electrostatic interactions occur in the (SSB)65 complexes formed with all poly-nucleotides, resulting in a net release of both cations and anions, although there are also contributions due to cation and anion uptake.

AB - The Escherichia coli single-strand binding (SSB) protein binds single-stranded (ss) nucleic acids in at least four distinct binding modes depending on the salt conditions [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799]. Equilibrium binding constants for the interaction of the E. coli SSB protein with poly(A), poly(U), poly(dA), and poly(dT) have been measured over a range of monovalent salt concentrations and types under conditions which favor only the high site size, (SSB)65 binding mode, which covers 65 nucleotides per SSB tetramer. The binding isotherms are analyzed by using a statistical thermodynamic model ("tetramer/octamer" model) that assumes cooperative binding of SSB is limited to the formation of octamers [Bujalowski, W., & Lohman, T. M. (1987) J. Mol. Biol. 195, 897] rather than the indefinite clustering of tetramers. The dependence of the intrinsic association equilibrium constant, Kobsd, and cooperatively parameter, ωT/O, on salt concentration has been determined by titrations which monitor the fluorescence quenching of the SSB protein upon complex formation. In the (SSB)65 binding mode, SSB binds with only moderate cooperativity to ss nucleic acids [Lohman, T. M., Overman, L. B., & Datta, S. (1986) J. Mol. Biol. 187, 603]. The cooperativity parameter derived from the tetramer/octamer model, which represents the equilibrium constant for formation of a nucleic acid bound SSB octamer from two nucleic acid bound tetramers, has a value of ωT/O = 410 ± 120 and is independent of salt concentration and type for poly(dA), poly(U), and poly(A) (25°C, pH 8.1). However, Kobsd decreases steeply with increasing salt concentration, such that ∂ log Kobsd/∂ log [NaCl] = -7.4 ± 0.5 for poly(U), -6.1 ± 0.6 for poly(dA), and -6.2 ± 0.3 for poly(A) (25.0°C, pH 8.1). The SSB-poly(dT) affinity is too high to measure in buffers containing even 5 M NaCl; however, in 1.8-2.5 M NaBr, we measure ∂ log Kobsd/∂ log [NaBr] = -5.7 ± 0.7, with a lower value of ωT/O = 130 ± 70. The polynucleotide specificity of the (SSB)65 binding mode (0.20 M NaCl, 25.0°C, pH 8.1) is Kobsd(dT) > Kobsd(dC) ≫ Kobsd(ss M13 DNA) > Kobsd(I) > Kobsd(U) = 8Kobsd(dA) = 87Kobsd(A) ≫ Kobsd(C). A dramatic effect of anion type on both the salt dependence and magnitude of Kobsd is also observed such that for poly(U) ∂ log Kobsd/∂ log [potassium glutamate] = -5.7 ± 0.4, ∂ log Kobsd/∂ log [NaF] = -4.3 ± 0.4, ∂ log Kobsd/∂ log [NaCH3COO] = -6.5 ± 0.2, and ∂ log Kobsd/∂ log [NaBr] = -6.7 ± 0.6; in 0.35 M monovalent cation, Kobsd(Glu) = 5.6Kobsd(F) = 11Kobsd(CH3COO) = (1.1 × 103)Kobsd(Cl) = (1.1 × 104)Kobsd(Br). These data indicate that significant electrostatic interactions occur in the (SSB)65 complexes formed with all poly-nucleotides, resulting in a net release of both cations and anions, although there are also contributions due to cation and anion uptake.

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EP - 471

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

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