A Sedimented Sample of a 59 kDa Dodecameric Helicase Yields High‐Resolution Solid‐State NMR Spectra

Gardiennet, Carole; Schütz, Anne K.; Hunkeler, Andreas; Kunert, Britta; Terradot, Laurent; Böckmann, Anja; Meier, Beat H.

doi:10.1002/anie.201200779

Cited by 117 publications

(125 citation statements)

References 32 publications

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“…The ultracentrifugation approach provides sufficient sensitivity for the study of large proteins, e.g., dodecamers of DnaB from Helicobacter pylori, representing a total mass of 708 kDa [50]. Figure 4 shows 2D NMR spectra of a crystalline (blue) and a sedimented (red) preparation.…”

Section: Sedimented Samples Produce Well-resolved Spectramentioning

confidence: 99%

Spinning proteins, the faster, the better?

Böckmann

Ernst

Meier

2015

Journal of Magnetic Resonance

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132

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Magic-angle spinning (MAS) is a technique that is a prerequisite for high-resolution solid-state NMR spectroscopy of proteins and other biomolecules. Recently, the 100 kHz limit for the rotation frequency has been broken, arguably making MAS rotors the man-made objects with the highest rotation frequency. This development is expected to have a significant impact on biomolecular NMR as it facilitates proton detection, which allows to partially compensate the loss in overall sensitivity associated with the small sample amounts that fit into MAS rotors with less than 1 mm outer diameter. Under these conditions, the mass-normalized sensitivity of a small rotor becomes much higher than that of larger-volume rotor.2

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Section: Sedimented Samples Produce Well-resolved Spectramentioning

confidence: 99%

Spinning proteins, the faster, the better?

Böckmann

Ernst

Meier

2015

Journal of Magnetic Resonance

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132

189

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“…The [1/2-13 C] Glc mixed sample was prepared in the same way by mixing [1-13 C] Glc labeled protein with [2-13 C] Glc labeled protein at an equimolar ratio. Approximately 15 mg MAVS CARD filament were packed into a 3.2-mm thin-wall NMR rotor by centrifugation at 100,000 × g for 15 h using a specially designed filling device (47). To probe the solvent-accessible surface of the MAVS CARD filament, 10 mL MAVS CARD filaments (1.5 mg/mL) was incubated overnight with 100 mM Gd-DTPA and then ultracentrifuged into an ssNMR rotor at 100,000 × g for 15 h.…”

Section: Methodsmentioning

confidence: 99%

Structure determination of helical filaments by solid-state NMR spectroscopy

Bardiaux

Ahmed

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The controlled formation of filamentous protein complexes plays a crucial role in many biological systems and represents an emerging paradigm in signal transduction. The mitochondrial antiviral signaling protein (MAVS) is a central signal transduction hub in innate immunity that is activated by a receptor-induced conversion into helical superstructures (filaments) assembled from its globular caspase activation and recruitment domain. Solid-state NMR (ssNMR) spectroscopy has become one of the most powerful techniques for atomic resolution structures of protein fibrils. However, for helical filaments, the determination of the correct symmetry parameters has remained a significant hurdle for any structural technique and could thus far not be precisely derived from ssNMR data. Here, we solved the atomic resolution structure of helical MAVS CARD filaments exclusively from ssNMR data. We present a generally applicable approach that systematically explores the helical symmetry space by efficient modeling of the helical structure restrained by interprotomer ssNMR distance restraints. Together with classical automated NMR structure calculation, this allowed us to faithfully determine the symmetry that defines the entire assembly. To validate our structure, we probed the protomer arrangement by solvent paramagnetic resonance enhancement, analysis of chemical shift differences relative to the solution NMR structure of the monomer, and mutagenesis. We provide detailed information on the atomic contacts that determine filament stability and describe mechanistic details on the formation of signaling-competent MAVS filaments from inactive monomers.solid-state NMR | protein structure | grid search | MAVS | innate immunity

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“…This manuscript presents the solid-state NMR spectroscopic investigation of the N-terminus of HpDnaB as a step towards an integrated structural biology approach aiming at a detailed structural description of the full-length protein. In this context, solidstate NMR studies on a sedimented sample of the fulllength protein have been described (Gardiennet et al 2012). Previous biochemical investigations suggest that the N-terminal domain and the linker region play an important role in multimerisation, quaternary state transition and activity of HpDnaB (Kashav et al 2009;Nitharwal et al 2007).…”

Section: Biological Contextmentioning

confidence: 99%

Solid-state NMR sequential assignments of the N-terminal domain of HpDnaB helicase

et al. 2015

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We present solid-state NMR assignments of the N-terminal domain of the DnaB helicase from Helicobacter pylori (153 residues) in its microcrystalline form. We use a sequential resonance assignment strategy based on three-dimensional NMR experiments. The resonance assignments obtained are compared with automated resonance assignments computed with the ssFLYA algorithm. An analysis of the (13)C secondary chemical shifts determines the position of the secondary structure elements in this α-helical protein.

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A Sedimented Sample of a 59 kDa Dodecameric Helicase Yields High‐Resolution Solid‐State NMR Spectra

Cited by 117 publications

References 32 publications

Spinning proteins, the faster, the better?

Spinning proteins, the faster, the better?

Structure determination of helical filaments by solid-state NMR spectroscopy

Solid-state NMR sequential assignments of the N-terminal domain of HpDnaB helicase

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