Spinning proteins, the faster, the better?

Böckmann, Anja; Ernst, Matthias; Meier, Beat H.

doi:10.1016/j.jmr.2015.01.012

Cited by 137 publications

(228 citation statements)

References 61 publications

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“…Reprotonation, e.g. by transaminases, has been observed before in wheat-germ cellfree expression (Tonelli et al 2011) and we are presently further investigating the reduction of the proton content which might indeed be the dominant reason for the observed line broadening (Böckmann et al 2015) leading to refocussable as well as not Hahn-echorefocussable contributions to T 2 relaxation. Another line to follow for obtaining narrower proton line widths might lie in the use of higher LPRs.…”

Section: Ns4b Lipid Reconstitution At Different Lprsmentioning

confidence: 55%

Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

et al. 2016

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Section: Ns4b Lipid Reconstitution At Different Lprsmentioning

confidence: 55%

Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

et al. 2016

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“…For the highly viscous fibrillar HET-s(218-289) sample prepared by sedimentation in an ultracentrifuge, the water component amounts to around 75% of the total sample mass as determined by the weight loss upon lyophilization. In general, upon application of MAS, which leads to centrifugation with over 10 6 g (Böckmann et al 2015) two water pools can be distinguished (Böckmann et al 2009): a supernatant pool in the center of the rotor, and a pool of water interacting with the protein. For the samples described here, the supernatant pool was minimized in mass by using a high centrifugation frequency and prolonged sedimentation times during rotor filling (see Materials and Methods).…”

Section: Spectroscopymentioning

confidence: 99%

Line-Broadening in Low-Temperature Solid-State NMR Spectra of Fibrils

et al. 2017

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Abstract:The temperature-dependent resonance-line broadening of in its amyloid form is investigated in the range between 110 K and 280 K. Significant differences are observed between residues in the structured hydrophobic triangular core, which are broadened the least and can be detected down to 100 K, and in the solvent-exposed parts, which are broadened the most and often disappear from the observed spectrum around 200 K. Below the freezing of the bulk water, around 273 K, the protein fibrils are still surrounded by a layer of mobile water whose thickness decreases with temperature, leading to drying out of the fibrils.

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“…High-resolution proton-detected methods were first demonstrated with modest spinning frequencies by today's standards (∼10 kHz) and relied on a reduction of 1 H-1 H couplings by high levels of dilution with deuterium, typically perdeuteration, and complete (8,9) or partial (10)(11)(12) protonation at exchangeable sites. The need for narrow proton resonances without such extreme levels of deuteration has motivated a continuous technological development, resulting in a dramatic increase in the available spinning frequency (13)(14)(15)(16)(17)(18)(19)(20).…”

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Structure of fully protonated proteins by proton-detected magic-angle spinning NMR

Andreas

Jaudzems

Staněk

et al. 2016

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

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Protein structure determination by proton-detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of signals from side chains is extremely limited. Here, we show in two fully protonated proteins that, at 100-kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of 1 H-1 H proximities that define a protein structure. The high data quality allowed the correct identification of internuclear distance restraints encoded in 3D spectra with automated data analysis, resulting in accurate, unbiased, and fast structure determination. Additionally, we find that narrower proton resonance lines, longer coherence lifetimes, and improved magnetization transfer offset the reduced sample size at 100-kHz spinning and above. Less than 2 weeks of experiment time and a single 0.5-mg sample was sufficient for the acquisition of all data necessary for backbone and side-chain resonance assignment and unsupervised structure determination. We expect the technique to pave the way for atomic-resolution structure analysis applicable to a wide range of proteins.NMR spectroscopy | magic-angle spinning | protein structures | proton detection | viral nucleocapsids D espite tremendous progress in the analysis of biomolecular samples over the last two decades (1-7), routine application of magic-angle spinning (MAS) NMR in biology is still limited by the inherently low sensitivity. The direct detection of proton resonances is a straightforward way to counter this problem, but entails a trade-off with resolution due to the strong homonuclear dipolar interactions among proton nuclei. High-resolution proton-detected methods were first demonstrated with modest spinning frequencies by today's standards (∼10 kHz) and relied on a reduction of 1 H-1 H couplings by high levels of dilution with deuterium, typically perdeuteration, and complete (8, 9) or partial (10-12) protonation at exchangeable sites. The need for narrow proton resonances without such extreme levels of deuteration has motivated a continuous technological development, resulting in a dramatic increase in the available spinning frequency (13)(14)(15)(16)(17)(18)(19)(20).At MAS frequencies of 40-60 kHz, deuteration and 100% reprotonation at exchangeable sites, primarily amide protons, result in resolved and sensitive spectra, similar in quality to the case of higher dilution levels and lower spinning frequencies (21-23). This opens the way to rapid sequential assignment of backbone resonances (24-27), as well as to the unambiguous measurement of detailed structural and dynamical parameters (28-32). A further increase in the MAS frequency to 100 kHz allows resonance assignment (20), a structure determination of a model protein (16), and interaction studies (15) with as little as 0.5 mg of sample. However, a high deuteration level severely limits observation of side-chain s...

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Spinning proteins, the faster, the better?

Cited by 137 publications

References 61 publications

Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

Line-Broadening in Low-Temperature Solid-State NMR Spectra of Fibrils

Structure of fully protonated proteins by proton-detected magic-angle spinning NMR

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