The bigger they are, the harder they fall: A topical review on sedimented solutes for solid‐state <scp>NMR</scp>

Ravera, Enrico

doi:10.1002/cmr.a.21318

Cited by 13 publications

(8 citation statements)

References 132 publications

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“…Herein, it is shown that highly resolved ssNMR spectra can be obtained for PEGylated proteins in the pelleted state, a densely packed non‐crystalline state of a wet macromolecular sample, which can be obtained either by rehydration of freeze‐dried material, or by ultracentrifugation when allowed by the density and molecular mass of the molecule . Such high‐quality ssNMR spectra are suitable for extensive resonance assignment and even conventional full structure determination.…”

Section: Figurementioning

confidence: 99%

Solid‐State NMR of PEGylated Proteins

et al. 2016

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PEGylated proteins are widely used in biomedicine but, in spite of their importance, no atomic-level information is available since they are generally resistant to structural characterization approaches. PEGylated proteins are shown here to yield highly resolved solid-state NMR spectra, which allows assessment of the structural integrity of proteins when PEGylated for therapeutic or diagnostic use.

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Section: Figurementioning

confidence: 99%

Solid‐State NMR of PEGylated Proteins

et al. 2016

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“…Using solid-state Nuclear Magnetic Resonance (NMR), a wide variety of biological materials can be studied, such as micro-or nanocrystals, fibrillar aggregates, and proteins embedded in membranes. Sedimentation of dissolved proteins from the solution state directly into the magic-angle spinning (MAS) rotor has lately become the most important sample preparation approach for solid-state NMR (Bertini et al, 2011(Bertini et al, , 2012(Bertini et al, , 2013Gardiennet et al, 2012;Ravera, 2014;van der Wel, 2018;Lacabanne et al, 2019a). This can be achieved in an ultracentrifuge (Böckmann et al, 2009;Bertini et al, 2012;Gardiennet et al, 2012) using specially designed rotor-filling tools (Böckmann et al, 2009;Mandal et al, 2017).…”

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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“…Concentrations can be pushed even further by desalting proteins and using labile buffers, such as ammonium formate or ammonium acetate, which are also removed by vacuum during the lyophilization process. This preparatory technique has been used particularly heavily in solid-state NMR studies to investigate protein structure, sedimentation and solvation effects, and even metabolic pathways in whole cells [27][28][29][30].…”

Section: Introductionmentioning

confidence: 99%

Preparation and spectroscopic characterization of lyophilized Mo nitrogenase

2020

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Mo nitrogenase is the primary source of biologically fixed nitrogen, making this system highly interesting for developing new, energy efficient ways of ammonia production. Although heavily investigated, studies of the active site of this enzyme have generally been limited to spectroscopic methods that are compatible with the presence of water and relatively low protein concentrations. One method of overcoming this limitation is through lyophilization, which allows for measurements to be performed on solvent free, high concentration samples. This method also has the potential for allowing efficient protein storage and solvent exchange. To investigate the viability of this preparatory method with Mo nitrogenase, we employ a combination of electron paramagnetic resonance, Mo and Fe K-edge X-ray absorption spectroscopy, and acetylene reduction assays. Our results show that while some small distortions in the metallocofactors occur, oxidation and spin states are maintained through the lyophilization process and that reconstitution of either lyophilized protein component into buffer restores acetylene reducing activity. Graphic abstract

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The bigger they are, the harder they fall: A topical review on sedimented solutes for solid‐state NMR

Cited by 13 publications

References 132 publications

Solid‐State NMR of PEGylated Proteins

Solid‐State NMR of PEGylated Proteins

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

Preparation and spectroscopic characterization of lyophilized Mo nitrogenase

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