The Experts below are selected from a list of 1221 Experts worldwide ranked by ideXlab platform
Beat H Meier - One of the best experts on this subject based on the ideXlab platform.
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ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter.
Molecules, 2020Co-Authors: Denis Lacabanne, Thomas Wiegand, Nino Wili, Maria I Kozlova, Riccardo Cadalbert, Daniel Klose, Armen Y Mulkidjanian, Beat H Meier, Anja BöckmannAbstract:Nucleoside triphosphates (NTPs) are used as chemical energy source in a variety of cell systems. Structural snapshots along the NTP hydrolysis reaction coordinate are typically obtained by adding stable, nonhydrolyzable adenosine triphosphate (ATP) -analogues to the proteins, with the goal to arrest a state that mimics as closely as possible a physiologically relevant state, e.g., the pre-hydrolytic, transition and post-hydrolytic states. We here present the lessons learned on two distinct ATPases on the best use and unexpected pitfalls observed for different analogues. The proteins investigated are the bacterial DnaB Helicase from Helicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis, both belonging to the same division of P-loop fold NTPases. We review the magnetic-resonance strategies which can be of use to probe the binding of the ATP-mimics, and present carbon-13, phosphorus-31, and vanadium-51 solid-state nuclear magnetic resonance (NMR) spectra of the proteins or the bound molecules to unravel conformational and dynamic changes upon binding of the ATP-mimics. Electron paramagnetic resonance (EPR), and in particular W-band electron-electron double resonance (ELDOR)-detected NMR, is of complementary use to assess binding of vanadate. We discuss which analogues best mimic the different hydrolysis states for the DnaB Helicase and the ABC transporter BmrA. These might be relevant also to structural and functional studies of other NTPases.
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High-spin Metal Centres in Dipolar EPR Spectroscopy.
CHIMIA International Journal for Chemistry, 2018Co-Authors: Katharina Keller, Thomas Wiegand, Riccardo Cadalbert, Beat H Meier, Anja Böckmann, Gunnar Jeschke, Maxim YulikovAbstract:The substitution of Mg2+ by Mn2+ in the bacterial DnaB Helicase from Helicobacter pylori, an ATP:Mg2+-fuelled protein engine, allows electron paramagnetic resonance (EPR) spectroscopy to be performed on this system. EPR experiments make it possible to monitor nucleotide binding and to estimate the fraction of bound Mn2+ through relaxation measurements. Furthermore, by measuring spin-spin distances we probe the geometry within such multimeric assemblies using ultra-wideband double electron-electron resonance (DEER) and relaxation induced dipolar modulation enhancement (RIDME). The extraction of distance distributions from RIDME experiments on high-spin paramagnetic centres is influenced by the presence of dipolar frequency overtones. We show herein that we can correct for these overtones by using a modified kernel function in Tikhonov regularization analysis routines, and that the overtone coefficients for Mn2+ in the DnaB Helicase are practically the same as in the previously studied Mn2+-Mn2+ model compounds.
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Protein–nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR
Journal of Biomolecular NMR, 2017Co-Authors: Thomas Wiegand, Riccardo Cadalbert, Anja Böckmann, Wei-chih Liao, Ta Chung Ong, Alexander Däpp, Christophe Copéret, Beat H MeierAbstract:DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein–DNA and protein–ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect protein–DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB Helicase from Helicobacter pylori complexed with ADP and single-stranded DNA, that this limitation can be overcome by using DNP-enhanced spectroscopy. Interactions are established by DNP-enhanced ^31P–^13C polarization-transfer experiments followed by the recording of a 2D ^13C–^13C correlation experiment. The NMR spectra were obtained in less than 2 days and allowed the identification of residues of the motor protein involved in nucleotide binding.
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Solid-state NMR and EPR Spectroscopy of Mn2+-Substituted ATP-Fueled Protein Engines
Angewandte Chemie International Edition, 2017Co-Authors: Thomas Wiegand, Denis Lacabanne, Riccardo Cadalbert, Beat H Meier, Laurent Terradot, Katharina Keller, Gunnar Jeschke, Maxim Yulikov, Lauriane Lecoq, Anja BöckmannAbstract:Paramagnetic metal ions deliver structural information both in EPR and solid-state NMR experiments, offering a profitable synergetic approach to study bio-macromolecules. We demonstrate the spectral consequences of Mg2+ / Mn2+ substitution and the resulting information contents for two different ATP:Mg2+ -fueled protein engines, a DnaB Helicase from Helicobacter pylori active in the bacterial replisome, and the ABC transporter BmrA, a bacterial efflux pump. We show that, while EPR spectra report on metal binding and provide information on the geometry of the metal centers in the proteins, paramagnetic relaxation enhancements identified in the NMR spectra can be used to localize residues at the binding site. Protein engines are ubiquitous and the methods described herein should be applicable in a broad context.
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Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid-State NMR Spectroscopy
Angewandte Chemie International Edition, 2016Co-Authors: Thomas Wiegand, Riccardo Cadalbert, Anja Böckmann, Carole Gardiennet, Joanna Timmins, Laurent Terradot, Beat H MeierAbstract:DnaB Helicases are bacterial, ATP-driven enzymes that unwind double-stranded DNA during DNA replication. Herein, we study the sequential binding of the "non-hydrolysable" ATP analogue AMP-PNP and of single-stranded (ss) DNA to the dodecameric DnaB Helicase from Helicobacter pylori using solid-state NMR. Phosphorus cross-polarization experiments monitor the binding of AMP-PNP and DNA to the Helicase. 13 C chemical-shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The Helicase switches upon AMP-PNP addition into a conformation apt for ssDNA binding, and AMP-PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP-PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid-state NMR can provide for the characterization of protein-DNA interactions and the interplay with ATP or its analogues.
Thomas Wiegand - One of the best experts on this subject based on the ideXlab platform.
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ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter.
Molecules, 2020Co-Authors: Denis Lacabanne, Thomas Wiegand, Nino Wili, Maria I Kozlova, Riccardo Cadalbert, Daniel Klose, Armen Y Mulkidjanian, Beat H Meier, Anja BöckmannAbstract:Nucleoside triphosphates (NTPs) are used as chemical energy source in a variety of cell systems. Structural snapshots along the NTP hydrolysis reaction coordinate are typically obtained by adding stable, nonhydrolyzable adenosine triphosphate (ATP) -analogues to the proteins, with the goal to arrest a state that mimics as closely as possible a physiologically relevant state, e.g., the pre-hydrolytic, transition and post-hydrolytic states. We here present the lessons learned on two distinct ATPases on the best use and unexpected pitfalls observed for different analogues. The proteins investigated are the bacterial DnaB Helicase from Helicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis, both belonging to the same division of P-loop fold NTPases. We review the magnetic-resonance strategies which can be of use to probe the binding of the ATP-mimics, and present carbon-13, phosphorus-31, and vanadium-51 solid-state nuclear magnetic resonance (NMR) spectra of the proteins or the bound molecules to unravel conformational and dynamic changes upon binding of the ATP-mimics. Electron paramagnetic resonance (EPR), and in particular W-band electron-electron double resonance (ELDOR)-detected NMR, is of complementary use to assess binding of vanadate. We discuss which analogues best mimic the different hydrolysis states for the DnaB Helicase and the ABC transporter BmrA. These might be relevant also to structural and functional studies of other NTPases.
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High-spin Metal Centres in Dipolar EPR Spectroscopy.
CHIMIA International Journal for Chemistry, 2018Co-Authors: Katharina Keller, Thomas Wiegand, Riccardo Cadalbert, Beat H Meier, Anja Böckmann, Gunnar Jeschke, Maxim YulikovAbstract:The substitution of Mg2+ by Mn2+ in the bacterial DnaB Helicase from Helicobacter pylori, an ATP:Mg2+-fuelled protein engine, allows electron paramagnetic resonance (EPR) spectroscopy to be performed on this system. EPR experiments make it possible to monitor nucleotide binding and to estimate the fraction of bound Mn2+ through relaxation measurements. Furthermore, by measuring spin-spin distances we probe the geometry within such multimeric assemblies using ultra-wideband double electron-electron resonance (DEER) and relaxation induced dipolar modulation enhancement (RIDME). The extraction of distance distributions from RIDME experiments on high-spin paramagnetic centres is influenced by the presence of dipolar frequency overtones. We show herein that we can correct for these overtones by using a modified kernel function in Tikhonov regularization analysis routines, and that the overtone coefficients for Mn2+ in the DnaB Helicase are practically the same as in the previously studied Mn2+-Mn2+ model compounds.
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Protein–nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR
Journal of Biomolecular NMR, 2017Co-Authors: Thomas Wiegand, Riccardo Cadalbert, Anja Böckmann, Wei-chih Liao, Ta Chung Ong, Alexander Däpp, Christophe Copéret, Beat H MeierAbstract:DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein–DNA and protein–ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect protein–DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB Helicase from Helicobacter pylori complexed with ADP and single-stranded DNA, that this limitation can be overcome by using DNP-enhanced spectroscopy. Interactions are established by DNP-enhanced ^31P–^13C polarization-transfer experiments followed by the recording of a 2D ^13C–^13C correlation experiment. The NMR spectra were obtained in less than 2 days and allowed the identification of residues of the motor protein involved in nucleotide binding.
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Solid-state NMR and EPR Spectroscopy of Mn2+-Substituted ATP-Fueled Protein Engines
Angewandte Chemie International Edition, 2017Co-Authors: Thomas Wiegand, Denis Lacabanne, Riccardo Cadalbert, Beat H Meier, Laurent Terradot, Katharina Keller, Gunnar Jeschke, Maxim Yulikov, Lauriane Lecoq, Anja BöckmannAbstract:Paramagnetic metal ions deliver structural information both in EPR and solid-state NMR experiments, offering a profitable synergetic approach to study bio-macromolecules. We demonstrate the spectral consequences of Mg2+ / Mn2+ substitution and the resulting information contents for two different ATP:Mg2+ -fueled protein engines, a DnaB Helicase from Helicobacter pylori active in the bacterial replisome, and the ABC transporter BmrA, a bacterial efflux pump. We show that, while EPR spectra report on metal binding and provide information on the geometry of the metal centers in the proteins, paramagnetic relaxation enhancements identified in the NMR spectra can be used to localize residues at the binding site. Protein engines are ubiquitous and the methods described herein should be applicable in a broad context.
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Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid-State NMR Spectroscopy
Angewandte Chemie International Edition, 2016Co-Authors: Thomas Wiegand, Riccardo Cadalbert, Anja Böckmann, Carole Gardiennet, Joanna Timmins, Laurent Terradot, Beat H MeierAbstract:DnaB Helicases are bacterial, ATP-driven enzymes that unwind double-stranded DNA during DNA replication. Herein, we study the sequential binding of the "non-hydrolysable" ATP analogue AMP-PNP and of single-stranded (ss) DNA to the dodecameric DnaB Helicase from Helicobacter pylori using solid-state NMR. Phosphorus cross-polarization experiments monitor the binding of AMP-PNP and DNA to the Helicase. 13 C chemical-shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The Helicase switches upon AMP-PNP addition into a conformation apt for ssDNA binding, and AMP-PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP-PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid-state NMR can provide for the characterization of protein-DNA interactions and the interplay with ATP or its analogues.
Subhasis B. Biswas - One of the best experts on this subject based on the ideXlab platform.
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An Essential DnaB Helicase of Bacillus anthracis: Identification, Characterization, and Mechanism of Action
Journal of Bacteriology, 2008Co-Authors: Esther E. Biswas, Marjorie H. Barnes, Donald T. Moir, Subhasis B. BiswasAbstract:We have described a novel essential replicative DNA Helicase from Bacillus anthracis, the identification of its gene, and the elucidation of its enzymatic characteristics. Anthrax DnaB Helicase (DnaBBA) is a 453-amino-acid, 50-kDa polypeptide with ATPase and DNA Helicase activities. DnaBBA displayed distinct enzymatic and kinetic properties. DnaBBA has low single-stranded DNA (ssDNA)-dependent ATPase activity but possesses a strong 5′→3′ DNA Helicase activity. The stimulation of ATPase activity appeared to be a function of the length of the ssDNA template rather than of ssDNA binding alone. The highest specific activity was observed with M13mp19 ssDNA. The results presented here indicated that the ATPase activity of DnaBBA was coupled to its migration on an ssDNA template rather than to DNA binding alone. It did not require nucleotide to bind ssDNA. DnaBBA demonstrated a strong DNA Helicase activity that required ATP or dATP. Therefore, DnaBBA has an attenuated ATPase activity and a highly active DNA Helicase activity. Based on the ratio of DNA Helicase and ATPase activities, DnaBBA is highly efficient in DNA unwinding and its coupling to ATP consumption.
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quantitative analysis of binding of single stranded dna by escherichia coli DnaB Helicase and the DnaB dnac complex
Biochemistry, 2006Co-Authors: Subhasis B. Biswas, Esther E BiswasfissAbstract:DnaB Helicase is responsible for unwinding duplex DNA during chromosomal DNA replication and is an essential component of the DNA replication apparatus in Escherichia coli. We have analyzed the mechanism of binding of single-stranded DNA (ssDNA) by the DnaB x DnaC complex and DnaB Helicase. Binding of ssDNA to DnaB Helicase was significantly modulated by nucleotide cofactors, and the modulation was distinctly different for its complex with DnaC. DnaB Helicase bound ssDNA with a high affinity [Kd = (5.09 +/- 0.32) x 10(-8) M] only in the presence of ATPgammaS, a nonhydrolyzable analogue of ATP, but not other nucleotides. The binding was sensitive to ionic strength but not to changes in temperature in the range of 30-37 degrees C. On the other hand, ssDNA binding in the presence of ADP was weaker than that observed with ATPgammaS, and the binding was insensitive to ionic strength. DnaC protein hexamerizes to form a 1:1 complex with the DnaB hexamer and loads it onto the ssDNA by forming a DnaB6 x DnaC6 dodecameric complex. Our results demonstrate that the DnaB6 x DnaC6 complex bound ssDNA with a high affinity [Kd = (6.26 +/- 0.65) x 10(-8) M] in the presence of ATP, unlike the DnaB hexamer. In the presence of ATPgammaS or ADP, binding of ssDNA by the DnaB6 x DnaC6 complex was a lower-affinity process. In summary, our results suggest that in the presence of ATP in vivo, the DnaB6 x DnaC6 complex should be more efficient in binding DNA as well as in loading DnaB onto the ssDNA than DnaB Helicase itself.
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Quantitative Analysis of Binding of Single-Stranded DNA by Escherichia coli DnaB Helicase and the DnaB·DnaC Complex†
Biochemistry, 2006Co-Authors: Subhasis B. Biswas, Esther E. Biswas-fissAbstract:DnaB Helicase is responsible for unwinding duplex DNA during chromosomal DNA replication and is an essential component of the DNA replication apparatus in Escherichia coli. We have analyzed the mechanism of binding of single-stranded DNA (ssDNA) by the DnaB x DnaC complex and DnaB Helicase. Binding of ssDNA to DnaB Helicase was significantly modulated by nucleotide cofactors, and the modulation was distinctly different for its complex with DnaC. DnaB Helicase bound ssDNA with a high affinity [Kd = (5.09 +/- 0.32) x 10(-8) M] only in the presence of ATPgammaS, a nonhydrolyzable analogue of ATP, but not other nucleotides. The binding was sensitive to ionic strength but not to changes in temperature in the range of 30-37 degrees C. On the other hand, ssDNA binding in the presence of ADP was weaker than that observed with ATPgammaS, and the binding was insensitive to ionic strength. DnaC protein hexamerizes to form a 1:1 complex with the DnaB hexamer and loads it onto the ssDNA by forming a DnaB6 x DnaC6 dodecameric complex. Our results demonstrate that the DnaB6 x DnaC6 complex bound ssDNA with a high affinity [Kd = (6.26 +/- 0.65) x 10(-8) M] in the presence of ATP, unlike the DnaB hexamer. In the presence of ATPgammaS or ADP, binding of ssDNA by the DnaB6 x DnaC6 complex was a lower-affinity process. In summary, our results suggest that in the presence of ATP in vivo, the DnaB6 x DnaC6 complex should be more efficient in binding DNA as well as in loading DnaB onto the ssDNA than DnaB Helicase itself.
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Mechanism and stoichiometry of interaction of DnaG primase with DnaB Helicase of Escherichia coli in RNA primer synthesis.
Journal of Biological Chemistry, 2003Co-Authors: Atanaska V. Mitkova, Sujata M. Khopde, Subhasis B. BiswasAbstract:Initiation and synthesis of RNA primers in the lagging strand of the replication fork in Escherichia coli requires the replicative DnaB Helicase and the DNA primase, the DnaG gene product. In addition, the physical interaction between these two replication enzymes appears to play a role in the initiation of chromosomal DNA replication. In vitro, DnaB Helicase stimulates primase to synthesize primers on single-stranded (ss) oligonucleotide templates. Earlier studies hypothesized that multiple primase molecules interact with each DnaB hexamer and single-stranded DNA. We have examined this hypothesis and determined the exact stoichiometry of primase to DnaB hexamer. We have also demonstrated that ssDNA binding activity of the DnaB Helicase is necessary for directing the primase to the initiator trinucleotide and synthesis of 11–20-nucleotide long primers. Although, association of these two enzymes determines the extent and rate of synthesis of the RNA primers in vitro, direct evidence of the formation of primase-DnaB complex has remained elusive in E. coli due to the transient nature of their interaction. Therefore, we stabilized this complex using a chemical crosslinker and carried out a stoichiometric analysis of this complex by gel filtration. This allowed us to demonstrate that the primase-Helicase complex of E. coli is comprised of three molecules of primase bound to one DnaB hexamer. Fluorescence anisotropy studies of the interaction of DnaB with primase, labeled with the fluorescent probe Ru(bipy)3, and Scatchard analysis further supported this conclusion. The addition of DnaC protein, leading to the formation of the DnaB-DnaC complex, to the simple priming system resulted in the synthesis of shorter primers. Therefore, interactions of the DnaB-primase complex with other replication factors might be critical for determining the physiological length of the RNA primers in vivo and the overall kinetics of primer synthesis.
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Conformational dynamics of DnaB Helicase upon DNA and nucleotide binding: analysis by intrinsic tryptophan fluorescence quenching.
Biochemistry, 2003Co-Authors: Stephen Flowers, Esther E. Biswas, Subhasis B. BiswasAbstract:DnaB Helicase of E. coli unwinds duplex DNA in the replication fork using the energy of ATP hydrolysis. We have analyzed structural and conformational changes in the DnaB protein in various nucleotides and DNA bound intermediate states by fluorescence quenching analysis of intrinsic fluorescence of native tryptophan (Trp) residues in DnaB. Fluorescence quenching analysis indicated that Trp48 in domain α is in a hydrophobic environment and resistant to fluorescence quenchers such as potassium iodide (KI). In domain β, Trp294 was found to be in a partially hydrophobic environment, whereas Trp456 in domain γ appeared to be in the least hydrophobic environment. Binding of oligonucleotides to DnaB Helicase resulted in a significant attenuation of the fluorescence quenching profile, indicating a change in conformation. ATPγS or ATP binding appeared to lead to a conformation in which Trp residues had a higher degree of solvent exposure and fluorescence quenching. However, the most dramatic increase of Trp fluore...
Anja Böckmann - One of the best experts on this subject based on the ideXlab platform.
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ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter.
Molecules, 2020Co-Authors: Denis Lacabanne, Thomas Wiegand, Nino Wili, Maria I Kozlova, Riccardo Cadalbert, Daniel Klose, Armen Y Mulkidjanian, Beat H Meier, Anja BöckmannAbstract:Nucleoside triphosphates (NTPs) are used as chemical energy source in a variety of cell systems. Structural snapshots along the NTP hydrolysis reaction coordinate are typically obtained by adding stable, nonhydrolyzable adenosine triphosphate (ATP) -analogues to the proteins, with the goal to arrest a state that mimics as closely as possible a physiologically relevant state, e.g., the pre-hydrolytic, transition and post-hydrolytic states. We here present the lessons learned on two distinct ATPases on the best use and unexpected pitfalls observed for different analogues. The proteins investigated are the bacterial DnaB Helicase from Helicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis, both belonging to the same division of P-loop fold NTPases. We review the magnetic-resonance strategies which can be of use to probe the binding of the ATP-mimics, and present carbon-13, phosphorus-31, and vanadium-51 solid-state nuclear magnetic resonance (NMR) spectra of the proteins or the bound molecules to unravel conformational and dynamic changes upon binding of the ATP-mimics. Electron paramagnetic resonance (EPR), and in particular W-band electron-electron double resonance (ELDOR)-detected NMR, is of complementary use to assess binding of vanadate. We discuss which analogues best mimic the different hydrolysis states for the DnaB Helicase and the ABC transporter BmrA. These might be relevant also to structural and functional studies of other NTPases.
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High-spin Metal Centres in Dipolar EPR Spectroscopy.
CHIMIA International Journal for Chemistry, 2018Co-Authors: Katharina Keller, Thomas Wiegand, Riccardo Cadalbert, Beat H Meier, Anja Böckmann, Gunnar Jeschke, Maxim YulikovAbstract:The substitution of Mg2+ by Mn2+ in the bacterial DnaB Helicase from Helicobacter pylori, an ATP:Mg2+-fuelled protein engine, allows electron paramagnetic resonance (EPR) spectroscopy to be performed on this system. EPR experiments make it possible to monitor nucleotide binding and to estimate the fraction of bound Mn2+ through relaxation measurements. Furthermore, by measuring spin-spin distances we probe the geometry within such multimeric assemblies using ultra-wideband double electron-electron resonance (DEER) and relaxation induced dipolar modulation enhancement (RIDME). The extraction of distance distributions from RIDME experiments on high-spin paramagnetic centres is influenced by the presence of dipolar frequency overtones. We show herein that we can correct for these overtones by using a modified kernel function in Tikhonov regularization analysis routines, and that the overtone coefficients for Mn2+ in the DnaB Helicase are practically the same as in the previously studied Mn2+-Mn2+ model compounds.
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Protein–nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR
Journal of Biomolecular NMR, 2017Co-Authors: Thomas Wiegand, Riccardo Cadalbert, Anja Böckmann, Wei-chih Liao, Ta Chung Ong, Alexander Däpp, Christophe Copéret, Beat H MeierAbstract:DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein–DNA and protein–ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect protein–DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB Helicase from Helicobacter pylori complexed with ADP and single-stranded DNA, that this limitation can be overcome by using DNP-enhanced spectroscopy. Interactions are established by DNP-enhanced ^31P–^13C polarization-transfer experiments followed by the recording of a 2D ^13C–^13C correlation experiment. The NMR spectra were obtained in less than 2 days and allowed the identification of residues of the motor protein involved in nucleotide binding.
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Solid-state NMR and EPR Spectroscopy of Mn2+-Substituted ATP-Fueled Protein Engines
Angewandte Chemie International Edition, 2017Co-Authors: Thomas Wiegand, Denis Lacabanne, Riccardo Cadalbert, Beat H Meier, Laurent Terradot, Katharina Keller, Gunnar Jeschke, Maxim Yulikov, Lauriane Lecoq, Anja BöckmannAbstract:Paramagnetic metal ions deliver structural information both in EPR and solid-state NMR experiments, offering a profitable synergetic approach to study bio-macromolecules. We demonstrate the spectral consequences of Mg2+ / Mn2+ substitution and the resulting information contents for two different ATP:Mg2+ -fueled protein engines, a DnaB Helicase from Helicobacter pylori active in the bacterial replisome, and the ABC transporter BmrA, a bacterial efflux pump. We show that, while EPR spectra report on metal binding and provide information on the geometry of the metal centers in the proteins, paramagnetic relaxation enhancements identified in the NMR spectra can be used to localize residues at the binding site. Protein engines are ubiquitous and the methods described herein should be applicable in a broad context.
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Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid-State NMR Spectroscopy
Angewandte Chemie International Edition, 2016Co-Authors: Thomas Wiegand, Riccardo Cadalbert, Anja Böckmann, Carole Gardiennet, Joanna Timmins, Laurent Terradot, Beat H MeierAbstract:DnaB Helicases are bacterial, ATP-driven enzymes that unwind double-stranded DNA during DNA replication. Herein, we study the sequential binding of the "non-hydrolysable" ATP analogue AMP-PNP and of single-stranded (ss) DNA to the dodecameric DnaB Helicase from Helicobacter pylori using solid-state NMR. Phosphorus cross-polarization experiments monitor the binding of AMP-PNP and DNA to the Helicase. 13 C chemical-shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The Helicase switches upon AMP-PNP addition into a conformation apt for ssDNA binding, and AMP-PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP-PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid-state NMR can provide for the characterization of protein-DNA interactions and the interplay with ATP or its analogues.
Riccardo Cadalbert - One of the best experts on this subject based on the ideXlab platform.
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ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter.
Molecules, 2020Co-Authors: Denis Lacabanne, Thomas Wiegand, Nino Wili, Maria I Kozlova, Riccardo Cadalbert, Daniel Klose, Armen Y Mulkidjanian, Beat H Meier, Anja BöckmannAbstract:Nucleoside triphosphates (NTPs) are used as chemical energy source in a variety of cell systems. Structural snapshots along the NTP hydrolysis reaction coordinate are typically obtained by adding stable, nonhydrolyzable adenosine triphosphate (ATP) -analogues to the proteins, with the goal to arrest a state that mimics as closely as possible a physiologically relevant state, e.g., the pre-hydrolytic, transition and post-hydrolytic states. We here present the lessons learned on two distinct ATPases on the best use and unexpected pitfalls observed for different analogues. The proteins investigated are the bacterial DnaB Helicase from Helicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis, both belonging to the same division of P-loop fold NTPases. We review the magnetic-resonance strategies which can be of use to probe the binding of the ATP-mimics, and present carbon-13, phosphorus-31, and vanadium-51 solid-state nuclear magnetic resonance (NMR) spectra of the proteins or the bound molecules to unravel conformational and dynamic changes upon binding of the ATP-mimics. Electron paramagnetic resonance (EPR), and in particular W-band electron-electron double resonance (ELDOR)-detected NMR, is of complementary use to assess binding of vanadate. We discuss which analogues best mimic the different hydrolysis states for the DnaB Helicase and the ABC transporter BmrA. These might be relevant also to structural and functional studies of other NTPases.
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High-spin Metal Centres in Dipolar EPR Spectroscopy.
CHIMIA International Journal for Chemistry, 2018Co-Authors: Katharina Keller, Thomas Wiegand, Riccardo Cadalbert, Beat H Meier, Anja Böckmann, Gunnar Jeschke, Maxim YulikovAbstract:The substitution of Mg2+ by Mn2+ in the bacterial DnaB Helicase from Helicobacter pylori, an ATP:Mg2+-fuelled protein engine, allows electron paramagnetic resonance (EPR) spectroscopy to be performed on this system. EPR experiments make it possible to monitor nucleotide binding and to estimate the fraction of bound Mn2+ through relaxation measurements. Furthermore, by measuring spin-spin distances we probe the geometry within such multimeric assemblies using ultra-wideband double electron-electron resonance (DEER) and relaxation induced dipolar modulation enhancement (RIDME). The extraction of distance distributions from RIDME experiments on high-spin paramagnetic centres is influenced by the presence of dipolar frequency overtones. We show herein that we can correct for these overtones by using a modified kernel function in Tikhonov regularization analysis routines, and that the overtone coefficients for Mn2+ in the DnaB Helicase are practically the same as in the previously studied Mn2+-Mn2+ model compounds.
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Protein–nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR
Journal of Biomolecular NMR, 2017Co-Authors: Thomas Wiegand, Riccardo Cadalbert, Anja Böckmann, Wei-chih Liao, Ta Chung Ong, Alexander Däpp, Christophe Copéret, Beat H MeierAbstract:DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein–DNA and protein–ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect protein–DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB Helicase from Helicobacter pylori complexed with ADP and single-stranded DNA, that this limitation can be overcome by using DNP-enhanced spectroscopy. Interactions are established by DNP-enhanced ^31P–^13C polarization-transfer experiments followed by the recording of a 2D ^13C–^13C correlation experiment. The NMR spectra were obtained in less than 2 days and allowed the identification of residues of the motor protein involved in nucleotide binding.
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Solid-state NMR and EPR Spectroscopy of Mn2+-Substituted ATP-Fueled Protein Engines
Angewandte Chemie International Edition, 2017Co-Authors: Thomas Wiegand, Denis Lacabanne, Riccardo Cadalbert, Beat H Meier, Laurent Terradot, Katharina Keller, Gunnar Jeschke, Maxim Yulikov, Lauriane Lecoq, Anja BöckmannAbstract:Paramagnetic metal ions deliver structural information both in EPR and solid-state NMR experiments, offering a profitable synergetic approach to study bio-macromolecules. We demonstrate the spectral consequences of Mg2+ / Mn2+ substitution and the resulting information contents for two different ATP:Mg2+ -fueled protein engines, a DnaB Helicase from Helicobacter pylori active in the bacterial replisome, and the ABC transporter BmrA, a bacterial efflux pump. We show that, while EPR spectra report on metal binding and provide information on the geometry of the metal centers in the proteins, paramagnetic relaxation enhancements identified in the NMR spectra can be used to localize residues at the binding site. Protein engines are ubiquitous and the methods described herein should be applicable in a broad context.
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Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid-State NMR Spectroscopy
Angewandte Chemie International Edition, 2016Co-Authors: Thomas Wiegand, Riccardo Cadalbert, Anja Böckmann, Carole Gardiennet, Joanna Timmins, Laurent Terradot, Beat H MeierAbstract:DnaB Helicases are bacterial, ATP-driven enzymes that unwind double-stranded DNA during DNA replication. Herein, we study the sequential binding of the "non-hydrolysable" ATP analogue AMP-PNP and of single-stranded (ss) DNA to the dodecameric DnaB Helicase from Helicobacter pylori using solid-state NMR. Phosphorus cross-polarization experiments monitor the binding of AMP-PNP and DNA to the Helicase. 13 C chemical-shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The Helicase switches upon AMP-PNP addition into a conformation apt for ssDNA binding, and AMP-PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP-PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid-state NMR can provide for the characterization of protein-DNA interactions and the interplay with ATP or its analogues.