Structural characteristics of the nucleotide-binding site of escherichia coli primary replicative helicase DnaB protein. studies with ribose and base-modified fluorescent nucleotide analogs

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Abstract

Structural characteristics of the base- and ribose-binding regions of the high-affinity noninteracting nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein have been studied, using the base-modified fluorescent nucleotide analog 1, N6-ethenoadenosine diphosphate (εADP) and the ribose-modified fluorescent analogs 3′(2′)-O-(N-methylantraniloyl)adenosine 5′-diphosphate (MANT-ADP), 3′-O-(N-methylantraniloyl)deoxyadenosine5′-diphosphate(MANT-dADP), 3′-O-(N-methylantraniloyl)-deoxyadenosine 5′-triphosphate (MANT-dATP), and 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-diphosphate (TNP-ADP). The obtained data indicate contrasting differences between these two regions. Binding of εADP to the DnaB helicase causes only ∼21% increase of the nucleotide fluorescence intensity and no shift of the emission spectrum maximum. The fluorescence of bound εADP is characterized by a single lifetime of 24.2 ± 0.6 ns, only slightly shorter than the fluorescent lifetime of the free εADP in solution (25.5 ± 0.6 ns). Solute-quenching studies of bound εADP, using different quenchers, acrylamide, I-, and Tl+, indicate limited accessibility of ethenoadenosine to the solvent. These results strongly suggest that the base-binding region of the DnaB nucleotide-binding site is located in the polar cleft on the enzyme's surface. Moreover, the limiting emission anisotropy of bound εADP is 0.21 ± 0.02, compared to the anisotropy of 0.3 of completely immobilized εADP at the same excitation wavelength (λex = 325 nm, λem = 410 nm), indicating that the adenine preserves substantial mobility when bound in the base-binding site. In contrast, fluorescence intensity at the emission maximum of TNP-ADP and MANT-ADP, which has modifying groups attached to the 2′ and/or 3′ oxygens of the ribose, increases upon binding to DnaB by factors of ∼4.7 (λex = 408 nm) and ∼2.6 (λex = 356 nm), respectively. Moreover, the maximum of emission spectrum of bound TNP-ADP is blue-shifted by ∼11 nm and that of MANT-ADP by ∼12 nm. Comparisons between spectral properties of TNP-ADP and MANT-ADP bound to DnaB and in different solvents suggest that the ribose-binding region of the DnaB nucleotide-binding site has relatively low polarity. Solute quenching studies of MANT-ADP fluorescence, using acrylamide, I-, and Tl+, indicate that the MANT group has very little accessibility to the solvent when bound to DnaB. Taken together, these results suggest that the ribose-binding region constitutes a hydrophobic cleft, or pocket, with very limited, if any, contact with the solvent. Moreover, fluorescence anisotropy of bound TNP-ADP and MANT-ADP is 0.32 ± 0.02 and 0.33 ± 0.02, respectively. These values are very close to the fundamental anisotropies of TNP-ADP and MANT-ADP, indicating that the fluorophores attached to the ribose have very restricted motional freedom. Fluorescence of MANT-ADP free in solution decays with a nearly homogenous single lifetime of 3.9 ± 0.2 ns. However, upon binding, the emission is characterized by two components (τ1 = 13.1 ± 0.5 ns, amplitude = 0.76, and τ2 = 6.0 ± 0.2 ns, amplitude = 0.24). Very similar double-exponential fluorescence decays have been obtained with bound MANT-dADP and MANT-dATP. The data indicate that the chromophores attached to the ribose experience two different environments when bound to the DnaB helicase, which may reflect the existence of two different conformations of the ribose-binding region of the DnaB helicase nucleotide-binding site. This result is in contrast to a single environment and probably a lack of discrete conformational heterogeneity at the base-binding region, as probed by εADP fluorescence. Thus, the data indicate that conformational heterogeneity is localized around the ribose and not transmitted to the base-binding region, suggesting limited communication between base- and ribose-binding sites. The reported results provide the first insight into the nature of the significant structural differences between base- and ribose-binding regions of the nucleotide-binding site of the DnaB helicase. The importance of these differences, in terms of the biological functioning of the DnaB protein, is discussed.

Original languageEnglish (US)
Pages (from-to)4682-4694
Number of pages13
JournalBiochemistry
Volume33
Issue number15
StatePublished - 1994

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DnaB Helicases
Ribose
Diphosphates
Adenosine Diphosphate
Escherichia coli
Adenosine
Nucleotides
Binding Sites
Proteins
Fluorescence
Anisotropy
Acrylamide
Quenching
Adenosine Diphosphate Ribose

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{2f7bfcca657e4ea98c92ccd8aa7a9a21,
title = "Structural characteristics of the nucleotide-binding site of escherichia coli primary replicative helicase DnaB protein. studies with ribose and base-modified fluorescent nucleotide analogs",
abstract = "Structural characteristics of the base- and ribose-binding regions of the high-affinity noninteracting nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein have been studied, using the base-modified fluorescent nucleotide analog 1, N6-ethenoadenosine diphosphate (εADP) and the ribose-modified fluorescent analogs 3′(2′)-O-(N-methylantraniloyl)adenosine 5′-diphosphate (MANT-ADP), 3′-O-(N-methylantraniloyl)deoxyadenosine5′-diphosphate(MANT-dADP), 3′-O-(N-methylantraniloyl)-deoxyadenosine 5′-triphosphate (MANT-dATP), and 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-diphosphate (TNP-ADP). The obtained data indicate contrasting differences between these two regions. Binding of εADP to the DnaB helicase causes only ∼21{\%} increase of the nucleotide fluorescence intensity and no shift of the emission spectrum maximum. The fluorescence of bound εADP is characterized by a single lifetime of 24.2 ± 0.6 ns, only slightly shorter than the fluorescent lifetime of the free εADP in solution (25.5 ± 0.6 ns). Solute-quenching studies of bound εADP, using different quenchers, acrylamide, I-, and Tl+, indicate limited accessibility of ethenoadenosine to the solvent. These results strongly suggest that the base-binding region of the DnaB nucleotide-binding site is located in the polar cleft on the enzyme's surface. Moreover, the limiting emission anisotropy of bound εADP is 0.21 ± 0.02, compared to the anisotropy of 0.3 of completely immobilized εADP at the same excitation wavelength (λex = 325 nm, λem = 410 nm), indicating that the adenine preserves substantial mobility when bound in the base-binding site. In contrast, fluorescence intensity at the emission maximum of TNP-ADP and MANT-ADP, which has modifying groups attached to the 2′ and/or 3′ oxygens of the ribose, increases upon binding to DnaB by factors of ∼4.7 (λex = 408 nm) and ∼2.6 (λex = 356 nm), respectively. Moreover, the maximum of emission spectrum of bound TNP-ADP is blue-shifted by ∼11 nm and that of MANT-ADP by ∼12 nm. Comparisons between spectral properties of TNP-ADP and MANT-ADP bound to DnaB and in different solvents suggest that the ribose-binding region of the DnaB nucleotide-binding site has relatively low polarity. Solute quenching studies of MANT-ADP fluorescence, using acrylamide, I-, and Tl+, indicate that the MANT group has very little accessibility to the solvent when bound to DnaB. Taken together, these results suggest that the ribose-binding region constitutes a hydrophobic cleft, or pocket, with very limited, if any, contact with the solvent. Moreover, fluorescence anisotropy of bound TNP-ADP and MANT-ADP is 0.32 ± 0.02 and 0.33 ± 0.02, respectively. These values are very close to the fundamental anisotropies of TNP-ADP and MANT-ADP, indicating that the fluorophores attached to the ribose have very restricted motional freedom. Fluorescence of MANT-ADP free in solution decays with a nearly homogenous single lifetime of 3.9 ± 0.2 ns. However, upon binding, the emission is characterized by two components (τ1 = 13.1 ± 0.5 ns, amplitude = 0.76, and τ2 = 6.0 ± 0.2 ns, amplitude = 0.24). Very similar double-exponential fluorescence decays have been obtained with bound MANT-dADP and MANT-dATP. The data indicate that the chromophores attached to the ribose experience two different environments when bound to the DnaB helicase, which may reflect the existence of two different conformations of the ribose-binding region of the DnaB helicase nucleotide-binding site. This result is in contrast to a single environment and probably a lack of discrete conformational heterogeneity at the base-binding region, as probed by εADP fluorescence. Thus, the data indicate that conformational heterogeneity is localized around the ribose and not transmitted to the base-binding region, suggesting limited communication between base- and ribose-binding sites. The reported results provide the first insight into the nature of the significant structural differences between base- and ribose-binding regions of the nucleotide-binding site of the DnaB helicase. The importance of these differences, in terms of the biological functioning of the DnaB protein, is discussed.",
author = "Wlodzimierz Bujalowski",
year = "1994",
language = "English (US)",
volume = "33",
pages = "4682--4694",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "15",

}

TY - JOUR

T1 - Structural characteristics of the nucleotide-binding site of escherichia coli primary replicative helicase DnaB protein. studies with ribose and base-modified fluorescent nucleotide analogs

AU - Bujalowski, Wlodzimierz

PY - 1994

Y1 - 1994

N2 - Structural characteristics of the base- and ribose-binding regions of the high-affinity noninteracting nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein have been studied, using the base-modified fluorescent nucleotide analog 1, N6-ethenoadenosine diphosphate (εADP) and the ribose-modified fluorescent analogs 3′(2′)-O-(N-methylantraniloyl)adenosine 5′-diphosphate (MANT-ADP), 3′-O-(N-methylantraniloyl)deoxyadenosine5′-diphosphate(MANT-dADP), 3′-O-(N-methylantraniloyl)-deoxyadenosine 5′-triphosphate (MANT-dATP), and 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-diphosphate (TNP-ADP). The obtained data indicate contrasting differences between these two regions. Binding of εADP to the DnaB helicase causes only ∼21% increase of the nucleotide fluorescence intensity and no shift of the emission spectrum maximum. The fluorescence of bound εADP is characterized by a single lifetime of 24.2 ± 0.6 ns, only slightly shorter than the fluorescent lifetime of the free εADP in solution (25.5 ± 0.6 ns). Solute-quenching studies of bound εADP, using different quenchers, acrylamide, I-, and Tl+, indicate limited accessibility of ethenoadenosine to the solvent. These results strongly suggest that the base-binding region of the DnaB nucleotide-binding site is located in the polar cleft on the enzyme's surface. Moreover, the limiting emission anisotropy of bound εADP is 0.21 ± 0.02, compared to the anisotropy of 0.3 of completely immobilized εADP at the same excitation wavelength (λex = 325 nm, λem = 410 nm), indicating that the adenine preserves substantial mobility when bound in the base-binding site. In contrast, fluorescence intensity at the emission maximum of TNP-ADP and MANT-ADP, which has modifying groups attached to the 2′ and/or 3′ oxygens of the ribose, increases upon binding to DnaB by factors of ∼4.7 (λex = 408 nm) and ∼2.6 (λex = 356 nm), respectively. Moreover, the maximum of emission spectrum of bound TNP-ADP is blue-shifted by ∼11 nm and that of MANT-ADP by ∼12 nm. Comparisons between spectral properties of TNP-ADP and MANT-ADP bound to DnaB and in different solvents suggest that the ribose-binding region of the DnaB nucleotide-binding site has relatively low polarity. Solute quenching studies of MANT-ADP fluorescence, using acrylamide, I-, and Tl+, indicate that the MANT group has very little accessibility to the solvent when bound to DnaB. Taken together, these results suggest that the ribose-binding region constitutes a hydrophobic cleft, or pocket, with very limited, if any, contact with the solvent. Moreover, fluorescence anisotropy of bound TNP-ADP and MANT-ADP is 0.32 ± 0.02 and 0.33 ± 0.02, respectively. These values are very close to the fundamental anisotropies of TNP-ADP and MANT-ADP, indicating that the fluorophores attached to the ribose have very restricted motional freedom. Fluorescence of MANT-ADP free in solution decays with a nearly homogenous single lifetime of 3.9 ± 0.2 ns. However, upon binding, the emission is characterized by two components (τ1 = 13.1 ± 0.5 ns, amplitude = 0.76, and τ2 = 6.0 ± 0.2 ns, amplitude = 0.24). Very similar double-exponential fluorescence decays have been obtained with bound MANT-dADP and MANT-dATP. The data indicate that the chromophores attached to the ribose experience two different environments when bound to the DnaB helicase, which may reflect the existence of two different conformations of the ribose-binding region of the DnaB helicase nucleotide-binding site. This result is in contrast to a single environment and probably a lack of discrete conformational heterogeneity at the base-binding region, as probed by εADP fluorescence. Thus, the data indicate that conformational heterogeneity is localized around the ribose and not transmitted to the base-binding region, suggesting limited communication between base- and ribose-binding sites. The reported results provide the first insight into the nature of the significant structural differences between base- and ribose-binding regions of the nucleotide-binding site of the DnaB helicase. The importance of these differences, in terms of the biological functioning of the DnaB protein, is discussed.

AB - Structural characteristics of the base- and ribose-binding regions of the high-affinity noninteracting nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein have been studied, using the base-modified fluorescent nucleotide analog 1, N6-ethenoadenosine diphosphate (εADP) and the ribose-modified fluorescent analogs 3′(2′)-O-(N-methylantraniloyl)adenosine 5′-diphosphate (MANT-ADP), 3′-O-(N-methylantraniloyl)deoxyadenosine5′-diphosphate(MANT-dADP), 3′-O-(N-methylantraniloyl)-deoxyadenosine 5′-triphosphate (MANT-dATP), and 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-diphosphate (TNP-ADP). The obtained data indicate contrasting differences between these two regions. Binding of εADP to the DnaB helicase causes only ∼21% increase of the nucleotide fluorescence intensity and no shift of the emission spectrum maximum. The fluorescence of bound εADP is characterized by a single lifetime of 24.2 ± 0.6 ns, only slightly shorter than the fluorescent lifetime of the free εADP in solution (25.5 ± 0.6 ns). Solute-quenching studies of bound εADP, using different quenchers, acrylamide, I-, and Tl+, indicate limited accessibility of ethenoadenosine to the solvent. These results strongly suggest that the base-binding region of the DnaB nucleotide-binding site is located in the polar cleft on the enzyme's surface. Moreover, the limiting emission anisotropy of bound εADP is 0.21 ± 0.02, compared to the anisotropy of 0.3 of completely immobilized εADP at the same excitation wavelength (λex = 325 nm, λem = 410 nm), indicating that the adenine preserves substantial mobility when bound in the base-binding site. In contrast, fluorescence intensity at the emission maximum of TNP-ADP and MANT-ADP, which has modifying groups attached to the 2′ and/or 3′ oxygens of the ribose, increases upon binding to DnaB by factors of ∼4.7 (λex = 408 nm) and ∼2.6 (λex = 356 nm), respectively. Moreover, the maximum of emission spectrum of bound TNP-ADP is blue-shifted by ∼11 nm and that of MANT-ADP by ∼12 nm. Comparisons between spectral properties of TNP-ADP and MANT-ADP bound to DnaB and in different solvents suggest that the ribose-binding region of the DnaB nucleotide-binding site has relatively low polarity. Solute quenching studies of MANT-ADP fluorescence, using acrylamide, I-, and Tl+, indicate that the MANT group has very little accessibility to the solvent when bound to DnaB. Taken together, these results suggest that the ribose-binding region constitutes a hydrophobic cleft, or pocket, with very limited, if any, contact with the solvent. Moreover, fluorescence anisotropy of bound TNP-ADP and MANT-ADP is 0.32 ± 0.02 and 0.33 ± 0.02, respectively. These values are very close to the fundamental anisotropies of TNP-ADP and MANT-ADP, indicating that the fluorophores attached to the ribose have very restricted motional freedom. Fluorescence of MANT-ADP free in solution decays with a nearly homogenous single lifetime of 3.9 ± 0.2 ns. However, upon binding, the emission is characterized by two components (τ1 = 13.1 ± 0.5 ns, amplitude = 0.76, and τ2 = 6.0 ± 0.2 ns, amplitude = 0.24). Very similar double-exponential fluorescence decays have been obtained with bound MANT-dADP and MANT-dATP. The data indicate that the chromophores attached to the ribose experience two different environments when bound to the DnaB helicase, which may reflect the existence of two different conformations of the ribose-binding region of the DnaB helicase nucleotide-binding site. This result is in contrast to a single environment and probably a lack of discrete conformational heterogeneity at the base-binding region, as probed by εADP fluorescence. Thus, the data indicate that conformational heterogeneity is localized around the ribose and not transmitted to the base-binding region, suggesting limited communication between base- and ribose-binding sites. The reported results provide the first insight into the nature of the significant structural differences between base- and ribose-binding regions of the nucleotide-binding site of the DnaB helicase. The importance of these differences, in terms of the biological functioning of the DnaB protein, is discussed.

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