Deuterated peptides and proteins in MAS solid-state NMR

Hologne, Maggy; Chevelkov, Veniamin; Reif, Bernd

doi:10.1016/j.pnmrs.2006.05.004

Cited by 77 publications

(94 citation statements)

References 149 publications

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“…Because amide proton chemical shifts are generally not observed in SSNMR (with notable exceptions) (Paulson et al 2003;Hologne et al 2006;Zhou et al 2007a,b), more accurate identification of amino acid types is required to efficiently link spin systems and map them back to the sequence. The iterative assignment process proceeds in stages.…”

Section: Resultsmentioning

confidence: 99%

Chemical shift assignment of the transmembrane helices of DsbB, a 20‐kDa integral membrane enzyme, by 3D magic‐angle spinning NMR spectroscopy

et al. 2008

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The Escherichia coli inner membrane enzyme DsbB catalyzes disulfide bond formation in periplasmic proteins, by transferring electrons to ubiquinone from DsbA, which in turn directly oxidizes cysteines in substrate proteins. We have previously shown that DsbB can be prepared in a state that gives highly resolved magic-angle spinning (MAS) NMR spectra. Here we report sequential 13 C and 15 N chemical shift assignments for the majority of the residues in the transmembrane helices, achieved by threedimensional (3D) correlation experiments on a uniformly 13 C, 15 N-labeled sample at 750-MHz 1 H frequency. We also present a four-dimensional (4D) correlation spectrum, which confirms assignments in some highly congested regions of the 3D spectra. Overall, our results show the potential to assign larger membrane proteins using 3D and 4D correlation experiments and form the basis of further structural and dynamical studies of DsbB by MAS NMR.

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Section: Resultsmentioning

confidence: 99%

Chemical shift assignment of the transmembrane helices of DsbB, a 20‐kDa integral membrane enzyme, by 3D magic‐angle spinning NMR spectroscopy

et al. 2008

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“…Obviously, the long-term goal should be to obtain model dependent information on dynamics. The quantification of the asymmetry parameter g of 2 H tensors (Hologne et al 2005(Hologne et al , 2006 or of dipolar coupling (Schanda et al 2011) allows to get information on motional models, such as jump angle and population of side chain rotameric states. Additional information might come from selectively side chain protonated samples in an otherwise deuterated matrix (Asami et al 2010;Asami and Reif 2013).…”

Section: Microcrystalline Proteinsmentioning

confidence: 99%

“…Large differential relaxation rates are found for D62, the N-terminus and the distal loop involving residues 45-50 a-spectrin SH3 12.4 ± 0.9 25.7 ± 1.8 25 ± 5 21.8 ± 3.3 14.6 ± 2.2 15 ± 3 T 2 was measured in an echo experiment with one 180°pulse in the center of the incremented delay for the H N bulk signal. The experiments were recorded at a magnetic field strength of 14.1 T and a MAS rotation frequency of 20 kHz and setting the effective temperature to 25°C deuterated samples, the spectral quality cannot be improved any further at a certain level of deuteration (Chevelkov et al 2006;Zhou et al 2007a, b;Akbey et al 2010;Lewandowski et al 2011a), indicating that dipolar interactions are not limiting spectral resolution. Similarly, spinning angle mis-adjustment results in an increase of the line width by a few Hertz (Chevelkov et al 2007b;Agarwal et al 2009).…”

Section: Limits Of Resolutionmentioning

confidence: 99%

Dynamics in the solid-state: perspectives for the investigation of amyloid aggregates, membrane proteins and soluble protein complexes

et al. 2014

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Aggregates formed by amyloidogenic peptides and proteins and reconstituted membrane protein preparations differ significantly in terms of the spectral quality that they display in solid-state NMR experiments. Structural heterogeneity and dynamics can both in principle account for that observation. This perspectives article aims to point out challenges and limitations, but also potential opportunities in the investigation of these systems.

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“…We and others could show previously, that deuteration and back-substitution of exchangeable protons allows for sensitive detection of protons [29][30][31], determination of long range 1 H, 1 H distances [32][33][34], and permits to localize mobile water molecules in the protein structure [35]. In addition, the 2 H spin can be used to probe side chain dynamics [36][37][38][39]. In contrast to the experimental scheme that was suggested by Ramamoorthy et al [40], use of high power decoupling is redundant using the deuteration approach.…”

Section: Introductionmentioning

confidence: 96%

Sensitivity enhancement using paramagnetic relaxation in MAS solid-state NMR of perdeuterated proteins

Linser

Chevelkov

Diehl

et al. 2007

Journal of Magnetic Resonance

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127

109

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Previously, Ishii et al., could show that chelated paramagnetic ions can be employed to significantly decrease the recycle delay of a MAS solid-state NMR experiment [N.P. Wickramasinghe, M. Kotecha, A. Samoson, J. Past, Y. Ishii, Sensitivity enhancement in C-13 solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing H-1 T-1 relaxation, J. Magn. Reson. 184 (2007) 350-356]. Application of the method is limited to very robust samples, for which sample stability is not compromised by RF induced heating. In addition, probe integrity might be perturbed in standard MAS PRE experiments due to the use of very short duty cycles. We show that these deleterious effects can be avoided if perdeuterated proteins are employed that have been re-crystallized from D 2 O:H 2 O = 9:1 containing buffer solutions. The experiments are demonstrated using the SH3 domain of chicken a-spectrin as a model system. The labeling scheme allows to record proton detected 1 H, 15 N correlation spectra with very high resolution in the absence of heteronuclear dipolar decoupling. Cu-edta as a doping reagent yields a reduction of the recycle delay by up to a factor of 15. In particular, we find that the 1 H T 1 for the bulk H N magnetization is reduced from 4.4 s to 0.3 s if the Cu-edta concentration is increased from 0 mM to 250 mM. Possible perturbations like chemical shift changes or line broadening due to the paramagnetic chelate complex are minimal. No degradation of our samples was observed in the course of the experiments.

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Deuterated peptides and proteins in MAS solid-state NMR

Cited by 77 publications

References 149 publications

Chemical shift assignment of the transmembrane helices of DsbB, a 20‐kDa integral membrane enzyme, by 3D magic‐angle spinning NMR spectroscopy

Chemical shift assignment of the transmembrane helices of DsbB, a 20‐kDa integral membrane enzyme, by 3D magic‐angle spinning NMR spectroscopy

Dynamics in the solid-state: perspectives for the investigation of amyloid aggregates, membrane proteins and soluble protein complexes

Sensitivity enhancement using paramagnetic relaxation in MAS solid-state NMR of perdeuterated proteins

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