Negative cooperativity in the binding of nucleotides to Escherichia coli replicative helicase DnaB protein. Interactions with fluorescent nucleotide analogs

Wlodzimierz Bujalowski, Malgorzata Maria Klonowska

Research output: Contribution to journalArticle

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Abstract

The interactions of nucleotides with Escherichia coli replicative helicase DnaB protein have been systematically studied using fluorescent nucleotide analogs, 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP), 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-diphosphate (TNP-ADP), 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-monophosphate (TNP-AMP), 3′-O-(N-methylantraniloyl) 5′-diphosphate (MANT-ADP), and 1,N6-ethenoadenosine diphosphate (εADP). The binding of the analogs is accompanied by strong quenching of the protein fluorescence; 0.76 ± 0.05, 0.76 ± 0.05, 0.58 ± 0.05, and 0.53 ± 0.5 for TNP-ATP, TNP-ADP, MANT-ADP, and εADP, respectively. A thermodynamically rigorous method has been applied to obtain all binding parameters from fluorescence titration curves independent of the assumption of strict proportionality between the observed quenching of the protein fluorescence and the degree of nucleotide binding. An exact representation of the observed fluorescence quenching, as a function of the nucleotide binding, is introduced through an empirical function which enables analysis of single binding isotherms without the necessity of determining all quenching constants for different binding sites. Using this method, we determined that, at saturation, the DnaB hexamer binds six molecules of TNP-ATP, TNP-ADP, MANT-ADP, and εADP, and that there is strong heterogeneity among nucleotide binding sites. The binding isotherms are biphasic. Three molecules of nucleotide are bound in the first high-affinity binding phase, and the subsequent three molecules are bound in the second low-affinity binding phase. The separation of the two binding steps is even more pronounced at higher temperatures. The change of the monitored fluorescence is sequential. The binding of the first nucleotide causes the largest quenching of the protein fluorescence with subsequent nucleotide binding inducing progressively less quenching. The simplest explanation of this behavior is that there is a negative cooperativity among nucleotide binding sites on a DnaB hexamer. The negative cooperativity is an intrinsic property of the DnaB helicase, since it is observed in the binding of nucleotide analogs which are different in type and location of the modifying group. A statistical thermodynamic model is proposed, the hexagon, which provides an excellent description of the binding process using only two interaction parameters, intrinsic binding constant K and cooperativity parameter σ. The data suggest an important role of the phosphate groups in binding and in recognition of nucleotides by the DnaB helicase.

Original languageEnglish (US)
Pages (from-to)5888-5900
Number of pages13
JournalBiochemistry
Volume32
Issue number22
StatePublished - 1993

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DnaB Helicases
Escherichia coli
Nucleotides
Adenosine
Adenosine Diphosphate
Quenching
Diphosphates
Fluorescence
Proteins
Adenosine Triphosphate
Binding Sites
Molecules
Isotherms
Statistical mechanics
Statistical Models
Adenosine Monophosphate
Titration
Thermodynamics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Negative cooperativity in the binding of nucleotides to Escherichia coli replicative helicase DnaB protein. Interactions with fluorescent nucleotide analogs. / Bujalowski, Wlodzimierz; Klonowska, Malgorzata Maria.

In: Biochemistry, Vol. 32, No. 22, 1993, p. 5888-5900.

Research output: Contribution to journalArticle

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N2 - The interactions of nucleotides with Escherichia coli replicative helicase DnaB protein have been systematically studied using fluorescent nucleotide analogs, 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP), 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-diphosphate (TNP-ADP), 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-monophosphate (TNP-AMP), 3′-O-(N-methylantraniloyl) 5′-diphosphate (MANT-ADP), and 1,N6-ethenoadenosine diphosphate (εADP). The binding of the analogs is accompanied by strong quenching of the protein fluorescence; 0.76 ± 0.05, 0.76 ± 0.05, 0.58 ± 0.05, and 0.53 ± 0.5 for TNP-ATP, TNP-ADP, MANT-ADP, and εADP, respectively. A thermodynamically rigorous method has been applied to obtain all binding parameters from fluorescence titration curves independent of the assumption of strict proportionality between the observed quenching of the protein fluorescence and the degree of nucleotide binding. An exact representation of the observed fluorescence quenching, as a function of the nucleotide binding, is introduced through an empirical function which enables analysis of single binding isotherms without the necessity of determining all quenching constants for different binding sites. Using this method, we determined that, at saturation, the DnaB hexamer binds six molecules of TNP-ATP, TNP-ADP, MANT-ADP, and εADP, and that there is strong heterogeneity among nucleotide binding sites. The binding isotherms are biphasic. Three molecules of nucleotide are bound in the first high-affinity binding phase, and the subsequent three molecules are bound in the second low-affinity binding phase. The separation of the two binding steps is even more pronounced at higher temperatures. The change of the monitored fluorescence is sequential. The binding of the first nucleotide causes the largest quenching of the protein fluorescence with subsequent nucleotide binding inducing progressively less quenching. The simplest explanation of this behavior is that there is a negative cooperativity among nucleotide binding sites on a DnaB hexamer. The negative cooperativity is an intrinsic property of the DnaB helicase, since it is observed in the binding of nucleotide analogs which are different in type and location of the modifying group. A statistical thermodynamic model is proposed, the hexagon, which provides an excellent description of the binding process using only two interaction parameters, intrinsic binding constant K and cooperativity parameter σ. The data suggest an important role of the phosphate groups in binding and in recognition of nucleotides by the DnaB helicase.

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