Solid-state NMR chemical-shift perturbations indicate domain reorientation of the DnaG primase in the primosome of Helicobacter pylori

Gardiennet, Carole; Wiegand, Thomas; Bazin, Alexandre; Cadalbert, Riccardo; Kunert, Britta; Lacabanne, Denis; Gutsche, Irina; Terradot, Laurent; Meier, Beat H.; Böckmann, Anja

doi:10.1007/s10858-016-0018-0

Cited by 14 publications

(19 citation statements)

References 21 publications

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“…The usefulness of particular ATP mimics for structural studies strongly depends on the nature of the protein of interest, as shown in Figure 2B for the example of DNA helicases and ABC transporters. The two proteins were subject to studies in the last years in our laboratories: the bacterial helicase DnaB from Helicobacter pylori [38,[80][81][82][83][84][85][86][87][88] and the ABC transporter BmrA from Bacillus subtilis [86,[89][90][91][92]. In the presence of double-stranded DNA, the DnaB from Helicobacter pylori is a double-homo hexamer of 59 kDa monomers with each hexamer moving along its single DNA strand, whereas BmrA from Bacillus subtilis is a dimeric membrane protein of 65 kDa monomers.…”

Section: The Dnab Helicase and The Abc Transporter Bmramentioning

confidence: 99%

ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter

et al. 2020

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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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Section: The Dnab Helicase and The Abc Transporter Bmramentioning

confidence: 99%

ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter

et al. 2020

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“…The detection of the paramagnetic effect was also performed with 3 control samples containing only the 0.7mM 15 N-labeled HBD, the DNA and the ATP bound protein, respectively. All studies have been conducted at 700 MHz, 298K with 90%:10% H 2 O/D 2 O. Solid-state NMR The protein-DNA complex solutions of pRN1 (40-370):ATP:DNA and pRN1-DNA were sedimented (Bertini et al, 2011;Gardiennet et al, 2012Gardiennet et al, , 2016 in thin-walled 3.2 mm MAS-NMR rotors (16 h at 4 C with 210000 g acceleration) using home-build tools (Bö ckmann et al, 2009). 13 C solid-state NMR spectra were acquired at 20.0 T static magnetic field strength using a 3.2 mm Bruker Biospin ' 'E-free'' probe (Gor'kov et al, 2007).…”

Section: Nmr Titrationsmentioning

confidence: 99%

A Small Helical Bundle Prepares Primer Synthesis by Binding Two Nucleotides that Enhance Sequence-Specific Recognition of the DNA Template

Boudet

Devillier

Wiegand

et al. 2019

Cell

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“…This is for some of these systems probably a consequence of oligomerization that has been induced by the high protein concentrations (Bertini et al, 2013;Fragai et al, 2013). Protein-protein complexes were studied by co-sedimentation (Gardiennet et al, 2016;Xiang et al, 2018) and protein-ligand complexes by sedimentation with the respective ligand (e.g. nucleic acids) directly into the NMR rotor (Wiegand et al, 2016a;Kaur et al, 2018;Stöppler et al, 2018;Lacabanne et al, 2019b).…”

Section: Introductionmentioning

confidence: 99%

Sedimentation Yields Long-Term Stable Protein Samples as Shown by Solid-State NMR

Wiegand

Lacabanne

Torosyan

et al. 2020

Front. Mol. Biosci.

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Today, the sedimentation of proteins into a magic-angle spinning (MAS) rotor gives access to fast and reliable sample preparation for solid-state Nuclear Magnetic Resonance (NMR), and this has allowed for the investigation of a variety of noncrystalline protein samples. High protein concentrations on the order of 400 mg/mL can be achieved, meaning that around 50-60% of the NMR rotor content is protein; the rest is a buffer solution, which includes counter ions to compensate for the charge of the protein. We have demonstrated herein the long-term stability of four sedimented proteins and complexes thereof with nucleotides, comprising a bacterial DnaB helicase, an ABC transporter, an archaeal primase, and an RNA polymerase subunit. Solid-state NMR spectra recorded directly after sample filling and up to 5 years later indicated no spectral differences and no loss in signal intensity, allowing us to conclude that protein sediments in the rotor can be stable over many years. We have illustrated, using an example of an ABC transporter, that not only the structure is maintained, but that the protein is still functional after long-term storage in the sedimented state.

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Solid-state NMR chemical-shift perturbations indicate domain reorientation of the DnaG primase in the primosome of Helicobacter pylori

Cited by 14 publications

References 21 publications

ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter

ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter

A Small Helical Bundle Prepares Primer Synthesis by Binding Two Nucleotides that Enhance Sequence-Specific Recognition of the DNA Template

Sedimentation Yields Long-Term Stable Protein Samples as Shown by Solid-State NMR

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